Methods and apparatus for characterization of polymers using multi-dimensional liquid chromatography with regular second-dimension sampling

ABSTRACT

Methods and apparatus for characterizing a polymer sample and in preferred embodiments, libraries of polymer samples, in a comprehensive, directly-coupled multi-dimensional liquid chromatography system are disclosed. The first and second dimensions are preferably high-performance liquid chromatography dimensions, such as for example, a first dimension adapted for determining composition (e.g. adapted for mobile-phase gradient elution chromatography, including reverse phase chromatography, adsorption chromatography and the like), and a second dimension adapted for determining molecular weight or particle size (e.g., adapted for size exclusion chromatography, including gel permeation chromatography).

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application hereby claims the benefit of co-pending U.S.application Ser. No. 60/315,685, entitled “Methods and Apparatus forCharacterization of Polymers Using Multi-Dimensional LiquidChromatography,” filed Aug. 28, 2001, the disclosure of which isincorporated by reference in its entirety.

BACKGROUND OF INVENTION

[0002] The present invention generally relates to methods and apparatusfor characterization of polymer samples in liquid chromatographysystems, and specifically, for characterization of polymer samples inmulti-dimensional liquid chromatography systems. The inventionparticularly relates, in a preferred embodiment, to characterization ofpolymer samples in a comprehensive, directly-coupled, multi-dimensionalhigh-performance liquid chromatography systems including a first HPLCdimension adapted for determining composition (e.g., adapted for reversephase chromatography, adsorption chromatography, and the like such asmobile phase gradient-elution chromatography) and a second HPLCdimension adapted for determining molecular weight or size (e.g.,adapted for size exclusion chromatography such as gel permeationchromatography).

[0003] Multi-dimensional high-performance liquid chromatography systemsare known in the art. See e.g., Murphy et al., Effect of Sampling Rateon Resolution in Comprehensive Two-Dimensional Liquid Chromatography,Anal. Chem. 70, 1585-1594 (1998); Murphy et al., One-and Two-DimensionalChromatographic Analysis of Alcohol Ethoxylates, Anal. Chem. 70,4353-4360 (1998); Kilz et al., Two Dimensional Chromatography for theDeformulation of Complex Copolymers, Chapter 17, pp. 223-241 of the textentitled “Chromatographic Characterization of Polymers, Hyphenated andMultidimensional Techniques”, edited by Provder et al. (AmericanChemical Society, Advances in Chemistry Series 247, 1995); Opiteck etal., Two-Dimensional SEC/RPLC Coupled to Mass Spectrometry for theAnalysis of Peptides, Anal. Chem. 69, 2283-2291 (1997); and Trathnigg etal., Two-Dimensional Liquid Chromatography of Functional Polyethers,Chapter 13, pp. 190-199 of the text entitled “Chromatography ofPolymers, Hyphenated and Multidimensional Techniques”, edited by Provderet al. (American Chemical Society, Symposium Series 731, 1999), each ofwhich is hereby incorporated by reference for all purposes.

[0004] Although the methods and systems disclosed to date in the arthave proven to be useful for characterizing biological andnon-biological polymer samples, they generally suffer frominefficiencies with respect to overall sample throughput, and/or withrespect to complicated control and/or operation schemes and systems.

[0005] Accordingly, there remains a need in the art for improved methodsand systems for effecting multi-dimensional liquid chromatography forcharacterization of polymer samples.

SUMMARY OF INVENTION

[0006] It is therefore an object of the present invention to providemethods and apparatus that allow for more efficient, and relatively lesscomplicated approaches than the prior art for characterizing polymersamples, and especially for fingerprinting polymer samples such asnon-biological copolymer samples.

[0007] Briefly, therefore, the present invention is directed, generally,to methods for characterizing a polymer sample in a multi-dimensionalliquid chromatography system. In preferred embodiments, the invention isdirected to methods for characterizing a library of polymer samples in amulti-dimensional liquid chromatography system. The multi-dimensionalliquid chromatography system comprises at least a first dimension and asecond dimension, and in some embodiments, can include a thirddimension, a fourth dimension and/or additional dimensions. Preferably,each of the first dimension and the second dimension is ahigh-performance liquid chromatography (HPLC) subsystem. Themulti-dimension liquid chromatography system is preferably acomprehensive multi-dimension liquid chromatography system wherein atleast a portion of each of the sample components separated in the firstdimension are further separated into subcomponents in the seconddimension. Further, the first dimension and second dimension of themulti-dimensional liquid chromatography system are preferablydirectly-coupled, wherein the components separated in the firstdimension are sampled in near real time (e.g., in-line) as they eluteoff of the first-dimension chromatography column(s) for injection intothe second dimension—for example, through a second-dimension in-linemulti-port injection valve.

[0008] The method generally comprises, for characterization of a singlepolymer sample, injecting the polymer sample into a first-dimensionhigh-performance liquid chromatography subsystem, separating the polymersample into two or more components in the first-dimension liquidchromatography subsystem, optionally detecting a property of thefirst-dimension separated components in the first-dimension eluent(e.g., using a flow-through detector), sampling at least a portion ofeach of the first-dimension separated components for directly-coupledinjection into a second dimension, injecting each of the sampledportions into a second-dimension high-performance liquid chromatographysubsystem, separating at least one of, and preferably each of thesampled portions of the first-dimension separated components into two ormore subcomponents in the second-dimension liquid chromatographysubsystem, and detecting a property of the second-dimension separatedsubcomponents in the second-dimension eluent (e.g., using a flow-throughdetector).

[0009] More specifically, for characterizing a single polymer sample,the polymer sample is injected (e.g., using a multi-port injection valveas a first-dimension injector) into a first-dimension mobile phase of afirst HPLC dimension of the multi-dimensional liquid chromatographysystem. At least one sample component of the injected polymer sample ischromatographically separated from other sample components thereof in afirst-dimension liquid chromatography column (e.g., in selectable fluidcommunication with the first-dimension injector), such that afirst-dimension mobile phase eluent from the first-dimension columncomprises two or more first-dimension separated sample components.Optionally, a property of the first-dimension separated components inthe first-dimension mobile phase effluent can be detected using aflow-through detector (e.g. mass detector, universal concentrationdetector, light-scattering detector, etc.). Then, at least a portion ofeach of the first-dimension separated sample components from thefirst-dimension mobile phase eluent are sampled for directly-coupledinjection into a second HPLC dimension of the multi-dimensionalchromatography system (e.g., using sample loops associated with amulti-port injection valve). The sampled portions of each of thefirst-dimension separated sample components are then injected directlyinto a second-dimension mobile phase of the second HPLC dimension of themulti-dimensional liquid chromatography system (e.g., using a multi-portinjection valve as a second-dimension injector). At least onesubcomponent of the injected sample portions is chromatographicallyseparated from other subcomponents thereof in a second-dimension liquidchromatography column (e.g., in selectable fluid communication with thesecond-dimension injector), such that a second-dimension mobile phaseeluent from the second-dimension column comprises two or moresecond-dimension separated subcomponents for one or more, and in somecases, for each of the sampled portions of each of the first-dimensionseparated sample components. A property of the second-dimensionseparated subcomponents are detected in the second-dimension mobilephase effluent using a flow-through detector.

[0010] For characterization of a library of polymer samples comprisingfour or more polymer samples, the aforementioned steps, as generally orspecifically characterized, of injecting into the first dimension,separating into components in the first dimension, optionally detectingseparated components in the first-dimension eluent, injecting into thesecond dimension, separating into subcomponents in the second dimensionand detecting separated subcomponents in the second-dimension eluent arerepeated for each of the polymer samples of the library.

[0011] In preferred embodiments, the method is further characterizedaccording to one or more of the following characterizing embodiments,considered independently or in combination in any of the variouspossible permutations.

[0012] In a first characterizing embodiment, at least a portion of eachof the first-dimension separated sample components are sampled byrepetitively sampling discrete volumes of the first-dimension mobilephase eluent at regularly recurring time intervals. That is, thesampling for the second dimension is effected at regular, recurringintervals of time without regard to whether or not a first-dimensionseparated component of the sample is present and actually sampled.Advantageously, such an approach is relatively less complicated thanother schemes for second-dimension sampling, is robust, and hasuniversal applicability across a wide range of polymers. Moreover, bycontrolling the separation rates of both the first and second dimensions(e.g., with the overall separation rate being characterized, forexample, as the injection rate into the first dimension), together withcontrolling the second-dimension sampling frequency and sample size,high-resolution multi-dimensional characterization can be effected.

[0013] In a second characterizing embodiment, the second-dimension ofthe multi-dimensional liquid chromatography system is a parallel-columnhigh-performance liquid chromatography subsystem, with serially-selectedor parallel detection. More specifically, the second dimension of themulti-dimensional liquid chromatography system comprises two or moreparallel second-dimension liquid chromatography columns, and asecond-dimension mobile phase is continuously supplied in parallelthrough the two or more second-dimension liquid chromatography columns.The sampled portions of the first-dimension separated sample componentsare serially and distributively injected into the second-dimensionmobile phases of the two or more second-dimension liquid chromatographycolumns, respectively. At least one subcomponent of the injected sampleportions is then chromatographically separated from other subcomponentsthereof substantially simultaneously (i.e., slightly offset temporally)in the respective second-dimension liquid chromatography columns.Advantageously, such an approach provides for substantially improvedoverall sample throughput, since the multiple second-dimension samplescan be substantially simultaneously evaluated, with a relativelyuncomplicated mechanical system comprising a single common injector.Moreover, effecting the chromatographic separation step of the seconddimension in parallel (i.e., substantially simultaneous separation usingtwo or more second-dimension columns) can advantageously provide asignificant improvement of the second dimension resolution by allowingfor relatively prolonged second dimension separation times for each ofthe sampled portions of the first-dimension eluent (as compared to astrictly serial second-dimension chromatographic separation andanalysis), while keeping the overall number of second dimensionseparations the same as can be effected in the serial second-dimensionseparation. Generally, the operational conditions of the first andsecond dimensions can be selected to achieve an appropriate balancebetween the overall sample throughput (in the first and/or seconddimension) and the desired resolution.

[0014] A third characterizing embodiment is directed to a method forcharacterizing a library of polymer samples. In this embodiment, alibrary of polymer samples are provided for characterization in themulti-dimensional liquid chromatography system, with the librarycomprising four or more different polymer samples for analysis. Themulti-dimensional liquid chromatography system comprises a firstdimension and a second dimension, with one of the first or seconddimensions being adapted for size exclusion chromatography. In aparticularly preferred embodiment, the second dimension HPLC subsystemis adapted for size-exclusion chromatography (SEC) such as gelpermeation chromatography (GPC). More specifically, in this thirdcharacterizing embodiment, at least a portion of each of thefirst-dimension separated sample components are sampled by sampling atleast ten discrete volumes of the first-dimension mobile phase eluent.The steps of injecting a polymer sample into the first-dimensionmobile-phase, chromatographically separating the injected polymer in thefirst dimension, optionally detecting a property of the first-dimensionseparated components, sampling the first-dimension mobile phase eluentfor injection into the second-dimension, injecting into the seconddimension, separating in the second dimension, and detecting a propertyof the second-dimension separated subcomponents are repeated for each ofthe four or more polymer samples of the library, with the four or morepolymer samples of the library being successively injected into thefirst-dimension mobile phase of the first dimension at intervals of notmore than about 30 minutes per sample. In preferred approaches for thisembodiment, the injection-to-injection interval is preferably not morethan about 15 minutes, and more preferably not more than about 10minutes.

[0015] The present invention is directed as well, to an apparatus foreffecting the above-identified methods. That is, the invention isdirected as well to multi-dimensional liquid chromatography systemscomprising a-first dimension high-performance liquid chromatographysubsystem and a second dimension high-performance liquid chromatographysubsystem. In general, the first dimension HPLC subsystem comprises afirst-dimension mobile phase source in fluid communication with afirst-dimension liquid chromatography column, a first-dimension pump influid communication with the first dimension mobile phase source andwith the first-dimension column for continuously supplying afirst-dimensional mobile phase through the first dimension column, aninjection valve in selectable fluid communication with thefirst-dimension mobile phase for serially injecting polymer samples intothe first-dimension mobile phase, and optionally, a first-dimensionflow-through detector in fluid communication with the first-dimensionmobile phase eluent for detecting a property of the first-dimensionseparated sample component. The second dimension HPLC subsystemcomprises a second-dimension mobile phase source in fluid communicationwith a second-dimension liquid chromatography column, a second-dimensionpump in fluid communication with the second dimension mobile phasesource and with the second-dimension column for continuously supplying asecond-dimensional mobile phase through the second dimension column, asecond-dimension injector in selectable fluid communication with thefirst-dimension mobile phase eluent and in selectable fluidcommunication with the second-dimension mobile phase for seriallysampling at least a portion of the first-dimension separated componentsfrom the first-dimension mobile phase eluent and for injecting thesampled portion into the second-dimension mobile phase, and asecond-dimension flow-through detector in fluid communication with thesecond-dimension mobile phase eluent for detecting a property of thesecond-dimension separated subcomponents.

[0016] In preferred embodiments, the multi-dimensional liquidchromatography systems are further characterized according to one ormore of the following characterizing embodiments, consideredindependently or in combination in any of the various possiblepermutations.

[0017] In one characterizing embodiment, the multi-dimensional liquidchromatography system is further characterized as comprising acontroller for the second-dimension injector, the controller beingadapted for sampling discrete volumes of the first-dimension mobilephase eluent at regularly recurring time intervals, and for injectingthe sampled volumes into the second-dimension mobile phase.

[0018] In another characterizing embodiment, the multi-dimensionalliquid chromatography system is further characterized as having afirst-dimension HPLC subsystem comprising a single mobile phase analysischannel, and a second-dimension HPLC subsystem comprising at least twoanalysis channels in parallel. More specifically, the second-dimensionHPLC subsystem comprises at least two second-dimension liquidchromatography columns, and is adapted to continuously supply thesecond-dimension mobile phase in parallel through the two or moresecond-dimension liquid chromatography columns (e.g., from thesecond-dimension mobile phase source). In preferred aspects of thischaracterizing embodiment, the second-dimension mobile-phase is suppliedto each of the second-dimension columns through one or more flowrestrictors.

[0019] In yet a further characterizing embodiment, the multi-dimensionalliquid chromatography system is further characterized as comprising acontrol system adapted for serially injecting successive polymer samplesinto the first dimension mobile phase of the system at intervals of notmore than about 30 minutes for sample, and adapted for sampling at leastten discrete volumes of the first-dimensional mobile phase eluent, andinjecting the at least ten sampled volumes directly into the seconddimension mobile phase.

[0020] In particularly preferred embodiments, including both methodembodiments and apparatus embodiments, the following features can beapplied generally with respect to any of the aforementioned embodiments,alone or in combination in the various permutations. Generally, thepolymer samples being characterized can be non-biological polymers(e.g., non-biological copolymers) or biological polymers (e.g.,proteins, DNA), and in many applications, are preferably non-biologicalpolymers. Generally, the first dimension HPLC subsystem can be adaptedfor chromatographic approaches effective for distinguishing betweenchemical composition and/or structural variations of polymer samplecomponents (e.g., repeat units types, ratios of copolymer repeat units,functional groups, branching, etc.). Exemplary preferred first-dimensionHPLC subsystems include reverse phase chromatography subsystems,mobile-phase compositional gradient elution chromatography subsystems,or mobile-phase temperature gradient elution chromatography subsystems.Mobile-phase elution gradients of the first dimension preferablycomprise a substantially universal co-solvent system, such as awater-tetrahydrofuran-hexane system. Generally, the second dimensionHPLC subsystem is preferably adapted for size-exclusion chromatography(SEC) such as gel permeation chromatography (GPC). Additionally, theflow-through detector of the second dimension HPLC subsystem isgenerally preferably a universal concentration detector or massdetector, such as an evaporative light-scattering detector (ELSD).Further, generally, the first and second dimension liquid chromatographysubsystems can be combined with further dimensions, such as third, forthor higher dimensions, and such further dimensions can beliquid-chromatography subsystems, gas-chromatography subsystems,electrophoretic subsystems, electrochromatographic subsystems,field-flow fractionation subsystems, flow-injection analysis subsystems,or other types of polymer characterization systems, such as massspectrometry.

[0021] The methods and apparatus of the invention are particularlyuseful for characterizing individual polymer samples, or libraries ofpolymer samples, such as non-biological polymer samples, and especiallyfor characterizing combinatorial libraries of polymers (e.g. synthesizedusing parallel polymerization approaches). The methods and apparatus ofthe invention can be advantageously applied for polymerfingerprinting—determining both compositional/structural characteristicsas well as molecular size/molecular weight characteristics. The methodsand apparatus of the invention can also be used for effective scale upof a polymerization synthesis process, to ensure that the fingerprint ofthe polymer made by large-scale synthesis process is substantially thesame as the polymer made by the smaller scale synthesis process.

[0022] The methods and apparatus of the invention can be applied usingconvention, macro-scale liquid chromatography systems, or alternatively,can be applied in a micro-scale or nano-scale format, such as inmicrofluidic devices such as lab-on-a-chip liquid microfluidicchromatography devices.

[0023] Other features, objects and advantages of the present inventionwill be in part apparent to those skilled in art and in part pointed outhereinafter. All references cited in the instant specification areincorporated by reference for all purposes. Moreover, as the patent andnon-patent literature relating to the subject matter disclosed and/orclaimed herein is substantial, many relevant references are available toa skilled artisan that will provide further instruction with respect tosuch subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a schematic representation of a multi-dimensional liquidchromatography system.

[0025]FIG. 2 is a schematic representation of a multi-port injectionvalve having two sample loops suitable for use as a second-dimensioninjector in the multi-dimensional liquid chromatography system of FIG.1.

[0026]FIGS. 3A and 3B are screen shots of graphical user interfaces thatallow for efficient user-driven control of hardware and data managementfunctions of a two-dimensional liquid chromatography system, as well asfor integrated display (FIG. 3A) or separate display (FIG. 3B) ofresulting characterization data.

[0027]FIGS. 4A and 4B are graphical data showing the results of amulti-dimensional liquid chromatography calibration. FIG. 4A is a graphshowing detector response (mV) versus retention time (min) with clearresolution of polystyrene standards subcomponents of different molecularweights. FIG. 4B is a graph showing the corresponding log molecularweigh data versus retention time (min) with the expected substantiallylinear relationship between components of the polystyrene standardssample.

[0028]FIGS. 5A and 5B are graphical data showing the results of amulti-dimensional liquid chromatography experiment. FIG. 5A is a3-dimensional plot showing detector response (V) versus both (i)normal-phase HPLC retention time (min), corresponding to thefirst-dimension separation, and (ii) GPC retention time (min),corresponding to the second-dimension separation, with clear resolutionof the various types of polymer components in the polymer sample. FIG.5B is a 2-dimensional contour graph showing the corresponding top-downview of the data presented in FIG. 5A, including normal-phase HPLCretention time (min), corresponding to the first-dimension separationversus GPC retention time (min), corresponding to the second-dimensionseparation, again showing clear resolution of the various types ofpolymer components.

[0029]FIG. 6 is a 2-dimensional graph showing the results of amulti-dimensional liquid chromatography experiment, includingspecifically, the relative normal-phase HPLC retention time,corresponding to the first-dimension separation, versus the relative GPCretention time (min), corresponding to the second-dimension separation.Clear resolution of the various types of polymer components in thepolymer sample is demonstrated.

[0030]FIGS. 7A through 7C are graphical representations of the librarydesign for a library of polymer samples (FIG. 7A), and the results of amulti-dimensional liquid chromatography experiment (FIGS. 7B and 7C).Specifically, FIG. 7B is a 3-dimensional plot showing molecular weight,as determined from second-dimension GPC data versus polystyrene standardcalibration, versus spatial position in the microtiter-format parallelreactor (columns 1-12 and rows 1-7). FIG. 7C is a 3-dimensional plotshowing chemical composition, as determined from first-dimension normalphase HPLC data (and shown as % of monomer B incorporated into each ofthe random copolymer samples), versus spatial position in themicrotiter-format parallel reactor (columns 1-12 and rows 1-7).

[0031]FIGS. 8A through 8C are graphical representations of the librarydesign for a library of polymer samples (FIG. 8A), the results of amulti-dimensional liquid chromatography experiment (FIG. 8B), and theoverlaid results of two separate, independent one-dimensional HPLCcharacterization experiments (FIG. 8C). Specifically, FIG. 8B is anarray of 2-dimensional contour graphs, each graph representing data fromone of the samples of the library, and each graph showing chemicalcomposition distribution (represented as normal phase HPLC retentiontime, corresponding to the relative amount of monomer B in each of thesamples), versus molecular weight distribution (represented as GPCretention time (-log MW). FIG. 8C shows the results of the independent,one-dimensional analysis for the same polymer samples for which data isshown in FIG. 8B, and is an array of 2-dimensional plots, each plotrepresenting the combined, independently-obtained data from one of thesamples of the library, and each plot showing chemical composition(represented as the relative amount of monomer B in each of the samplesas determined by the independent, one-dimension normal phase HPLCgradient elution characterization), versus molecular weight (representedas GPC log MW.

[0032]FIGS. 9A and 9B are plots showing data from a characterization ofa polymer sample using two-dimensional chromatography (FIG. 9A) andusing a conventional GPC-FTIR techniques (FIG. 9B). FIG. 9A is a2-dimensional contour graph showing normal-phase HPLC retention time(sec), corresponding to the first-dimension separation, versus GPCretention time (min), corresponding to the second-dimension separation,with clear resolution of the polymer components. FIG. 9B is a plotshowing the data from the GPC-FTIR.

[0033]FIGS. 10A and 10B are 2-dimensional contour graphs showingchemical composition distribution, represented by polarity (asdetermined using normal-phase HPLC retention time in a first-dimensionanalysis), versus molecular weight distribution (as determined using GPCretention time in a second-dimension analysis) for a small-scale sampleof interest (FIG. 10A) and for the corresponding scaled-up sample ofinterest (FIG. 10B).

DETAILED DESCRIPTION OF THE INVENTION

[0034] In the present invention, methods and apparatus are disclosed forcharacterization of single polymer samples and/or for characterizationof a library comprising four or more polymer samples. Preferably, thecharacterization methods and apparatus can be applied for fingerprintingbiological and non-biological polymer samples—for analysis in ananalytical laboratory, or for analysis in an on-line, near real timeprocess monitoring or process control system.

[0035] Certain characterizing features of this invention are related toinventions described in co-owned U.S. patent applications, including (i)U.S. Ser. No. 09/710,801, (now U.S. Pat. No. 6,406,632), entitled “RapidCharacterization of Polymers,” filed Nov. 8, 2000 by Safir et al., as acontinuation application of U.S. Ser. No. 09/285,363, filed Apr. 2, 1999(now abandoned), which itself claimed the benefit of U.S. provisionalapplication Serial No. 60/080,652, filed Apr. 3, 1998 (the applicationSer. No. 09/710,801 being hereinafter referred to as the “Co-Owned RapidCharacterization of Polymers Application”), and (ii) U.S. Ser. No.09/410,546, (now U.S. Pat. No. 6,296,771), entitled “ParallelHigh-Performance Liquid Chromatography with Serial Injection,” filedOct. 1, 1999 by Petro et al, as a continuation-in-part application ofthe following applications: U.S. Ser. No. 09/285,363, U.S. Ser. No.09/285,393 (now U.S. Pat. No. 6,265,226), U.S. Ser. No. 09/285,333 (nowU.S. Pat. No. 6,260,407), U.S. Ser. No. 09/285,335 (now U.S. Pat. No.6,175,409), and U.S. Ser. No. 09/285,392 (now U.S. Pat. No. 6,294,388),each of which themselves claimed the benefit of U.S. provisionalapplication Serial No. 60/080,652, filed Apr. 3, 1998 (the Ser. No.09/410,546 being hereinafter referred to as the “Co-Owned Parallel HPLCApplication”). Each of the aforementioned co-owned U.S. patentapplications (i.e., the Co-Owned Rapid Characterization of PolymersApplication and the Co-Owned Parallel HPLC Application) are herebyincorporated by reference for all purposes. Many features of the presentinvention will be described hereinafter with reference to the Co-OwnedRapid Characterization of Polymers Application and/or the Co-OwnedParallel HPLC Application.

[0036] The invention is described in further detail below with referenceto the figures, in which like items are numbered the same in the severalfigures.

[0037] A multi-dimensional liquid chromatography system of the presentinvention comprises a first dimension liquid chromatography subsystem,and a second dimension liquid chromatography subsystem, and optionally,third dimension and/or fourth dimension and/or additional dimensionsubsystems. Although the first dimension and second dimension are liquidchromatography subsystems, and preferably high-performance liquidchromatography subsystems, the additional dimension subsystems can beliquid chromatography subsystems, gas chromatography subsystems,electrophoretic subsystems, electrochromatographic subsystems,field-flow fractionation subsystems, flow-injection analysis subsystemsor other types of polymer characterization subsystems, including forexample, mass spectrometry.

[0038] Referring to FIG. 1, the multi-dimensional liquid chromatographysystem 10 can include a first dimension HPLC subsystem 1000 and a seconddimension HPLC subsystem 2000. In general, the first dimension HPLCsubsystem 1000 comprises a first-dimension mobile phase source (e.g., asshown, in one or more mobile phase reservoirs) 1100 a, 1100 b in fluidcommunication with a first-dimension liquid chromatography column 1500.A first-dimension pump 1200 provides fluid communication between thefirst dimension mobile phase source 1100 a, 1100 b and with thefirst-dimension column 1500 for continuously supplying afirst-dimensional mobile phase through the first dimension column 1500.The first dimension HPLC subsystem 1000 further comprises an injectionvalve 1300, that can include an injection port 1310 for receivingpolymer samples from a sample source (e.g., such as a sample handlingrobot 1410 of polymer sampling system 1400, or from an on-line samplingsystem in a polymerization process line, not shown). The first-dimensioninjection valve 1300 is in selectable fluid communication with thefirst-dimension mobile phase for serially injecting polymer samples 100into the first-dimension mobile phase. Although not shown in FIG. 1, thefirst dimension HPLC subsystem can optionally further include afirst-dimension flow-through detector in fluid communication with thefirst-dimension mobile phase eluent for detecting a property of thefirst-dimension separated sample component. First-dimension mobile phaseeluent is, as discussed in further detail below, discharged through asecond-dimension sampling and injection system (generally referred toherein as a second-dimension injector) and/or to an exhaust or wasteport or collection reservoir.

[0039] With further reference to FIG. 1, the second dimension HPLCsubsystem 2000 comprises a second-dimension mobile phase source 2100 influid communication with a second-dimension liquid chromatography column2500. A second-dimension pump 2200 provides fluid communication betweenthe second dimension mobile phase source 2100 and with thesecond-dimension column 2500 for continuously supplying asecond-dimensional mobile phase through the second dimension column2500. A second-dimension injector 2300 is in selectable fluidcommunication with the first-dimension mobile phase eluent for seriallysampling at least a portion of the first-dimension separated componentsfrom the first-dimension mobile phase eluent. The sampling is generallyeffected in discrete volumes, as further discussed below. Thesecond-dimension injector 2300 is also in selectable fluid communicationwith the second-dimension mobile phase for injecting the sampled portion(e.g., the discrete sampled volumes taken from the first-dimensionmobile phase eluent) into the second-dimension mobile phase. Thesecond-dimension injector 2300 can also include additional hardware,such as flow-splitters, for changing the concentration and/or flow rateof the sampled portion of the first-dimension eluent. The seconddimension HPLC subsystem further comprises one or more second-dimensionflow-through detectors 2600 in fluid communication with thesecond-dimension mobile phase eluent for detecting a property of thesecond-dimension separated subcomponents.

[0040] The first dimension HPLC subsystem 1000 and the second dimensionHPLC subsystem 2000 of the multi-dimensional liquid chromatographysystem 10 are preferably directly-coupled, wherein components of thepolymer sample 100 separated in the first dimension are sampled in nearreal time (e.g., in-line) from the first-dimension eluent as they eluteoff of the first-dimension chromatography column(s). The sampledfirst-dimension separated components (or one or more portions thereof)are then injected into the second dimension—for example, through asecond-dimension injector 2300. Preferably, the second-dimensioninjector 2300 is an integral second-dimension injector 2300 that isfunctionally a component of the both the first-dimension subsystem 1000and the second-dimension subsystem 2000—and is adapted for both samplingand injecting. The sampling and injection functions could, however, beaccomplished using non-integral components, provided as separate systemcomponents, and linked for example, manually or using robotic transfer(not shown). Such a non-integral second-dimension sampling and injectionsystem is still considered to be directly coupled, provided that thereis no long term storage of the sampled portions of the first-dimensioneluent prior to injection into the second-dimension eluent. Preferably,the discrete volumes sampled from the first dimension eluent are notstored at all, and are injected immediately, in sequential steady stateoperation, into the mobile phase of the second dimension. It is,nonetheless, contemplated that some built-in time delay could beincorporated into the method to allow for treatment of the sampledportion prior to injection into the second-dimension mobile phase. Forexample, sampled portions of the first-dimension mobile phase eluentcould be stored (e.g., for treatment or otherwise) for not more thanabout 4 hours, preferably not more than about 2 hours, more preferablystill not more than about 1 hour, and still more preferably not morethan about 30 minutes, 10 minutes, 5 minutes, 2 minutes, 1 minute, 30seconds, 15 seconds, 10 seconds or 5 seconds. As noted, thesecond-dimension injector 2300 could also include additionalfunctionality, such as flow-splitting for changing the concentrationand/or flow rate of the sampled portion of the first-dimension eluent.

[0041] Referring now to FIG. 2, in many embodiments, thesecond-dimension injector 2300 can be a multi-port injection valve2300′, preferably comprising one or more sample loops 2310, 2320.Operation of such multi-port injection valves are discussed in detail inthe Rapid Characterization of Polymers Application. Briefly, in a firstswitch position and valve configuration depicted in FIG. 2, the firstdimension mobile phase eluent is discharged into an inlet port 2330 ofthe injection valve 2300′. The first switch position and valveconfiguration allows for the first-dimension eluent to pass through ainlet 2321 of the sample loop 2320, through the sample loop 2320, andthrough an outlet 2323 thereof to waste, thereby loading the sample loop2320. Meanwhile, the second-dimension mobile phase is being routedthrough the other sample loop 2310 to the second dimension column 2500.When the switch position and valve configuration is switched to analternative second switch position and valve configuration (not shown),the inlet 2321 of the sample loop 2320 is aligned with the seconddimension mobile phase coming from the second dimension pump 2200, andthe outlet 2323 of the sample loop 2320 is aligned for fluidcommunication with the second-dimension column 2500, such that adiscrete volume sample equal to the volume of the sample loop 2320 isinjected into the second-dimension mobile phase. Repeated alternationbetween the first and second switch positions/valve configurationsallows for alternating loading of sample loops 2310, 2320 and injectingof corresponding discrete sample volumes into the second dimensionmobile phase.

[0042] Other types of injection valves can be used, including forexample, arrays of microvalves configured for sampling and injection,for example, analogous to that described in co-pending U.S. patentapplication Ser. No. 60/274,022 entitled “Gas Chromatograph InjectionValve Having Microvalve Array” filed Mar. 7, 2001 by Bergh et al, whichis hereby incorporated by reference for all purposes.

[0043] In operation, with further reference to FIGS. 1 and 2, forcharacterization of a single polymer sample, a polymer sample 100 isinjected into a first-dimension high-performance liquid chromatographysubsystem 1000 through first dimension injector 1300. The polymer sample100 is separated into two or more components in the first-dimensionliquid chromatography column 1500, with the two or more components beingdischarged from the column 1500 as part of the first dimension mobilephase eluent. Optionally, a property of the first-dimension separatedcomponents in the first-dimension eluent can be detected (e.g., using aflow-through detector). At least a portion of the first-dimensionseparated components are sampled, for example, using thesecond-dimension injector 2300 for directly-coupled injection into thesecond dimension HPLC subsystem 2000. The sampled portions of thefirst-dimension separated components are separated into two or moresubcomponents in the second-dimension liquid chromatography columns2500, and discharged therefrom as part of the second-dimension mobilephase eluent. A property of the second-dimension separated subcomponentsis detected in the second-dimension eluent (e.g., using a flow-throughdetector 2600). For characterization of a library of polymer samplescomprising four or more polymer samples, the aforementioned steps ofinjecting into the first dimension, separating into components in thefirst dimension, optionally detecting separated components in thefirst-dimension eluent, injecting into the second dimension, separatinginto subcomponents in the second dimension and detecting separatedsubcomponents in the second-dimension eluent are repeated for each ofthe polymer samples of the library.

[0044] The multi-dimension liquid chromatography system is preferably acomprehensive multi-dimension liquid chromatography system wherein atleast a portion of each of the sample components separated in the firstdimension are further separated into subcomponents in the seconddimension. Preferably, the separation rates of the first dimension, theseparation rates of the second dimension, the sampling interval (i.e.,sampling frequency) for sampling of the first-dimension mobile phaseeluent and/or the sampling volume of the sampled portions offirst-dimension mobile phase eluent are controlled, independently, invarious combinations and/or in combination with other factors, such thatat least two discrete fractions of each of the first-dimension separatedsample components are sampled. More preferably, such factors arecontrolled such that at least three discrete fractions, and in someapplications, even higher numbers of discrete fractions such as at leastfour discrete fractions, at least five discrete fractions or at leastsix discrete fractions of each of the first-dimensions separated samplecomponents are sampled.

[0045] Certain preferred characterizing embodiments of the invention aredescribed as follows. These embodiments can be applied individually, orin various combinations, including each of the various permutationsthereof. Moreover, certain more general features of the invention, thatcan be commonly applied to each of these preferred characterizingembodiments or the various possible combinations thereof, are alsodescribed hereinafter. As noted, reference is made as appropriate to theaforementioned co-owned related applications, namely the Co-Owned RapidCharacterization of Polymers Application and the Co-Owned Parallel HPLCApplication.

[0046] Regularly-Recurring 2nd Dimension Sampling Interval

[0047] In one preferred embodiment, at least a portion of each of thefirst-dimension separated sample components are sampled by repetitivelysampling discrete volumes of the first-dimension mobile phase eluent atregularly recurring time intervals. That is, the sampling for the seconddimension is effected at regular, recurring intervals of time withoutregard to whether or not a first-dimension separated component of thesample is present and actually sampled. Hence, the multi-dimensionalliquid chromatography system can be further characterized as comprisingone or more controllers, including for example, controllers forcontrolling the separation rate of each of the first dimension andsecond dimension, and especially in particular, a controller for thesecond-dimension injector, the second-dimension injector controllerbeing adapted for sampling discrete volumes of the first-dimensionmobile phase eluent at regularly recurring time intervals, and forinjecting the sampled volumes into the second-dimension mobile phase.

[0048] In general, the time interval that defines the sampling frequencyfor sampling the first-dimension mobile phase eluent, and preferably,that also defines the injection frequency of the sampled portion intothe second-dimension mobile phase, is not narrowly critical, and canrange, for example, from about 10 minutes to about 5 seconds or less.Preferably, the time interval that defines the sampling frequency canrange from about 5 minutes to about 10 seconds, and in some embodiments,from about 2 minutes to about 30 seconds. Generally, therefore, adiscrete volume of the first-dimension mobile phase is sampled (andpreferably, also injected into the second-dimension mobile phase) atleast once every 10 minutes, and more preferably at least once every 5minutes, and most preferably at least once every 2 minutes. In someembodiments, a discrete volume of the first-dimension mobile phase canbe sampled (and preferably, also injected into the second-dimensionmobile phase) at least once every 180 seconds, and more preferably atleast once every 1. minute, even more preferably at least once every 30seconds, and in some cases, at least once every 15 seconds, at leastonce every 10 seconds or at least once every 5 seconds.

[0049] The sampled volume from the first-dimension mobile phase eluentis likewise not narrowly critical, and can vary depending on the natureand/or goals of the analysis. In preferred applications, for example,the sampled volume can range from about 5 ml to about 5 μl, preferablyfrom about 1 ml to about 10 μl and more preferably from about 500 μl toabout 25 μl. Generally, therefore, the sampled volumes offirst-dimension mobile phase eluent are preferably not more than about 5ml, preferably not more than about 1 ml, and more preferably not morethan about 500 μl. In some embodiments, the sampled volumes offirst-dimension mobile phase eluent can be not more than about 250 μl,not more than about 100 μl, not more than about 50 μl, not more thanabout 25 μl, not more than about 10 μl, or not more than about 5 μl, ornot more than about 1 μl. Such smaller volume samples forsecond-dimension characterization can have applications, for example, inmicro-scale and nano-scale multi-dimensional chromatography systems,such as lab-on-a-chip type systems.

[0050] The absolute number of discrete sample volumes sampled from thefirst-dimension mobile phase eluent for injection into thesecond-dimension mobile phase can, for each polymer sample beingcharacterized, vary widely depending on the nature and/or goals of theanalysis. In many embodiments, for example, the number of discretevolumes of the first-dimension eluent that are sampled (for each polymersample being characterized) can range from about 5 to about 5000,preferably from about 10 to about 1000, and more preferably from about20 to about 500, and in some embodiments, from about 100 to about 400.Generally, therefore, the number of discrete volumes of the of thefirst-dimension eluent that are sampled (for each polymer sample beingcharacterized) is preferably at least about 5, more preferably at leastabout 10, even more preferably at least about 20, and in someembodiments, at least about 50, at least about 100, at least about 200,at least about 400, at least about 500, at least about 1000, or at leastabout 5000 or more.

[0051] In a particularly preferred approach of this preferredembodiment—in which at least a portion of each of the first-dimensionseparated sample components are sampled by repetitively samplingdiscrete volumes of the first-dimension mobile phase eluent at regularlyrecurring time intervals—the sampling frequency from the first-dimensionmobile phase eluent, and/or the injection frequency to thesecond-dimension, together with the sampled volumes can be controlled,in combination, such that substantially all of the first-dimensionmobile phase eluent coming off of the first-dimension column is sampledand subsequently injected into a second-dimension mobile phase forsecond-dimension analysis. Preferably, the amount of the first-dimensionmobile phase eluent coming off of the first-dimension column that issampled and subsequently injected into a second-dimension mobile phasefor second-dimension analysis is at least about 70% or more, and in someembodiments, can be at least about 80%, at least about 90%, at leastabout 95%, at least about 97% or at least about 99%. Such an approachcan substantially approximate a completely-coupled, continuously-coupledand directly-coupled multi-dimensional liquid chromatography system. Ina preferred embodiment, for example, at least ten discrete volumes ofthe first-dimension mobile phase eluent are sampled with a samplingfrequency of at least once every 30 seconds, with the sampled volumesbeing not more than about 250 μl. If for example, the first-dimensionmobile phase flow rate in such a system is about 0.5 ml/min, and thesampling frequency in this system is, in fact, about once every thirtyseconds, with a sampled volume of about 250 μl, then all of thefirst-dimension mobile-phase eluent is sampled and preferably alsoinjected into the second dimension mobile phase. As another example,with the same first dimension flow rate (0.5 ml/min) and samplingfrequency (2 times/minute), if the sampled volume were only about 200μl, then about 80% of the total first-dimension mobile phase eluentvolume would be sampled into the second dimension. As a further example,with the same first dimension flow rate (0.5 ml/min), but with a highersampling frequency (4 times/minute), and with a smaller sampled volume(about 100 μl), then about 80% of the total first-dimension mobile phaseeluent volume would again be sampled into the second dimension.

[0052] In the directly-coupled, regularly recurring sampling embodimentsdescribed above, particularly where the first dimension and seconddimension are completely, and continuously coupled (e.g., withrelatively small sample volumes), at least one of the sampled volumes ofthe first-dimension mobile phase eluent may consist essentially of thefirst-dimension mobile phase, and have an essential absence offirst-dimension separated sample components.

[0053] 2^(nd)-Dimension Analysis with Parallel ChromatographicSeparation

[0054] In another preferred embodiment, the second-dimension of themulti-dimensional liquid chromatography system is adapted for parallel,or at least substantially parallel chromatographic separation of thesampled portions of the first-dimension separated components. That is,the second-dimension subsystem of the invention can be a parallel-columnhigh-performance liquid chromatography subsystem, preferably with asingle, common second-dimension injector. More specifically, in thispreferred embodiment, the multi-dimensional liquid chromatography systemincludes a first-dimension HPLC subsystem comprising a single mobilephase analysis channel, and a second-dimension HPLC subsystem comprisingat least two analysis channels in parallel, preferably directly coupledthrough a single, common second-dimension injector. The second dimensionHPLC subsystem comprises two or more parallel second-dimension liquidchromatography columns adapted such that a second-dimension mobile phaseis continuously supplied in parallel through the two or moresecond-dimension columns. (e.g., from the second-dimension mobile phasesource). In one embodiment, a second-dimension mobile phase can becontinuously supplied in parallel to the two or more second-dimensionliquid chromatography columns through two or more supply conduits, eachof the two or more supply conduits providing continuous parallel fluidcommunication between a second-dimension liquid mobile-phase source andthe two or more second-dimension liquid chromatography columns. Inpreferred aspects of this embodiment, the fluid communication path toeach of the two or more second-dimension columns includes one or moreflow restrictors associated with each supply conduit. Further detailsregarding the flow restrictors, and other aspects of this embodiment aresubstantially as described in the Co-Owned Parallel HPLC PatentApplication.

[0055] In a particularly preferred approach for this embodiment—anapproach employing a single, common second-dimension injector forcoupling the first and second dimension—the sampled portions of thefirst-dimension separated sample components are serially anddistributively injected into the second-dimension mobile phases of thetwo or more second-dimension liquid chromatography columns,respectively. At least one subcomponent of the injected sample portionsis then chromatographically separated from other subcomponents thereofsubstantially simultaneously (i.e., slightly offset temporally) in therespective second-dimension liquid chromatography columns (as comparedbetween second-dimension columns).

[0056] Coupled sampling between the first-dimension and second-dimensionsubsystems can be effected, as described above, such that a portion ofthe first-dimension separated components are sampled for injection intothe second-dimension mobile phases at regularly recurring timeintervals. In an alternative approach, however, the coupling can also bea controlled coupling. Specifically, a portion of the first-dimensionseparated components can be sampled for injection into thesecond-dimension mobile phases at intervals triggered by a controlsignal based on detection of the first-dimension separated components inthe first-dimension mobile phase eluent.

[0057] Detection in the second dimension can generally be effectedserially (e.g., with a selection valve for directing the two or moresecond-dimension mobile phase eluents to a detector) or in parallel. Inparallel second-dimension detection embodiments, each of the two or morechromatographic columns can have its own dedicated detector, such thatdetection of subcomponents derived from different sampled portions ofthe first-dimension eluent occurs substantially simultaneously ascompared between different analysis channels of the second dimension.For any given sampled component (or portion thereof) of thefirst-dimension, however, once injected into a particular analysischannel of the second dimension, detection of properties of thesecond-dimension separated subcomponents is effected serially withinthat analysis channel. The second dimension detector is preferably anoptical detector. An optical detector can be advantageous applied,particularly in highly parallel systems, and/or in systems designed tobe effective for nano-scale and/or micro-scale analysis (e.g.,lab-on-a-chip applications). An optical detector, such as alight-scattering detector or other optical detector, can be applieddirectly to samples that can be detected by the optical detector. Insome cases, however, the detectability of the sampled separatedsubcomponents can be developed, for example, by treatingsecond-dimension separated subcomponents to change an optical propertythereof before detection with an optical detector.

[0058] The number of parallel second-dimension chromatographic columns,and associated second-dimension mobile phases is not of crucialsignificance, but is preferably four or more second-dimensionchromatographic columns, and more preferably eight or moresecond-dimension chromatographic columns. Higher numbers can also beemployed, as described for example in the Co-Owned Parallel HPLCApplication.

[0059] In one preferred characterization protocol of this embodiment,injection into the second-dimension mobile phase is effected by asecond-dimension injection system comprising the second-dimensioninjector and a multi-port switching valve. The injector has asample-loading port for serially receiving a plurality of sampledportions and has a sample-discharge port for discharging the pluralityof sampled portions under pressure to the switching valve. The switchingvalve can have an inlet port and two or more selectable outlet ports,the inlet port being in fluid communication with the sample-dischargeport of the injector and being in selectable fluid communication withthe two or more selectable outlet ports, the two or more selectableoutlet ports being in fluid communication with the two or moresecond-dimension chromatography columns, respectively, such that thesampled portions can be serially and distributively injected into thesecond-dimension mobile phases of the two or more second-dimensionliquid chromatography columns. In a particularly preferred variation ofthis preferred protocol, in which at least ten portions of thefirst-dimension mobile phase eluent are sampled, the second-dimensioninjector is a multi-port switching valve having at least two sampleloops, and the second-dimension multi-port switching valve is controlledsuch that a first sampled portion is injected into a mobile phase of thefirst column, a second sampled portion is injected into a mobile phaseof the second column, a third sampled portion is injected into a mobilephase of the third column, a fourth sampled portion is injected into amobile phase of the fourth column, a fifth sampled portion is injectedinto a mobile phase of the first column, a sixth sampled portion isinjected into a mobile phase of the second column, a seventh sampledportion is injected into a mobile phase of the third column, an eighthsampled portion is injected into a mobile phase of the fourth column, aninth sampled portion is injected into a mobile phase of the firstcolumn, and a tenth sampled portion is injected into a mobile phase ofthe second column.

[0060] In another preferred protocol, especially where the seconddimension of the multi-dimensional liquid chromatography systemscomprises four or more parallel second-dimension liquid chromatographycolumns, the method can be directed to characterizing a provided librarycomprising ten or more different polymer samples, where the polymersamples are polymerization product mixtures resulting frompolymerization reactions that are varied with respect to reactionconditions, reactants, catalysts, catalyst precursors, initiators,additives or the relative amounts thereof. The ten or more polymersamples are serially injected into the first-dimension mobile phasethrough a first-dimension injector, and a continuously supplied mobilephase is provided in parallel through the four or more second-dimensionliquid chromatography columns. The sampled portions of thefirst-dimension separated components of ten or more polymer samples areserially and distributively injected into the second-dimension mobilephases of the four or more chromatographic columns through a commonsecond-dimension injector.

[0061] The number of sampled volumes for second-dimension analysis, thevolume thereof, and the second-dimension sampling frequency can be thesame as described above in connection with the regularly-recurringsecond-dimension injection interval embodiment.

[0062] Further detailed description of the second-dimension parallelHPLC subsystem, both apparatus and operational aspects thereof is setforth below, in the Co-Owned Parallel HPLC Application.

[0063] High-Throughput 2-Dimensional Chromatography with One SECDimension

[0064] A further preferred embodiment is directed to a method forcharacterizing a library of polymer samples. In this embodiment, alibrary of polymer samples are provided for characterization in themulti-dimensional liquid chromatography system, with the librarycomprising four or more different polymer samples for analysis. Themulti-dimensional liquid chromatography system comprises a firstdimension and a second dimension, with one of the first or seconddimensions being adapted for size exclusion chromatography. In aparticularly preferred embodiment, the second dimension HPLC subsystemis adapted for size-exclusion chromatography (SEC) such as gelpermeation chromatography (GPC).

[0065] More specifically, in this embodiment, at least a portion of eachof the first-dimension separated sample components are sampled bysampling at least ten discrete volumes of the first-dimension mobilephase eluent. The steps of injecting a polymer sample into thefirst-dimension mobile-phase, chromatographically separating theinjected polymer in the first dimension, optionally detecting a propertyof the first-dimension separated components, sampling thefirst-dimension mobile phase eluent for injection into thesecond-dimension, injecting into the second dimension, separating in thesecond dimension, and detecting a property of the second-dimensionseparated subcomponents are repeated for each of the four or morepolymer samples of the library, with the four or more polymer samples ofthe library being successively injected into the first-dimension mobilephase of the first dimension at intervals of not more than about 30minutes per sample.

[0066] In preferred approaches for this embodiment, the first-dimensioninjection-to-injection interval is preferably not more than about 15minutes, more preferably not more than about 10 minutes, and mostpreferably not more than about 8 minutes per sample. In someembodiments, the overall throughput of the two-dimensionalchromatography system, as characterized by first-dimensioninjection-to-injection interval, can be not more than about 4 minutes,not more than about 2 minutes, not more than about 1 minute and/or notmore than about 30 seconds per sample.

[0067] The number of sampled volumes for second-dimension analysis, thevolume thereof, and the second-dimension sampling frequency can be thesame as described above in connection with the regularly-recurringsecond-dimension injection interval embodiment. Additionally, coupledsampling between the first-dimension and second-dimension subsystems canbe effected, as described above, such that a portion of thefirst-dimension separated components are sampled for injection into thesecond-dimension mobile phases at regularly recurring time intervals. Inan alternative approach, however, the coupling can also be a controlledcoupling. Specifically, a portion of the first-dimension separatedcomponents can be sampled for injection into the second-dimension mobilephases at intervals triggered by a control signal based on detection ofthe first-dimension separated components in the first-dimension mobilephase eluent. Moreover, the second dimension can have a single analysischannel, or can comprise parallel analysis channels, as described above.

[0068] Further details of this high-throughput embodiment are describedas set forth in the Co-Owned Rapid Characterization of PolymersApplication.

[0069] General Features and Protocols

[0070] The following features and protocols are general to each of theaforementioned embodiments, and can be applied generally thereto, andused in combination generally therewith.

[0071] Generally, the polymer samples being characterized can benon-biological polymers (e.g., non-biological copolymers) or biologicalpolymers (e.g., proteins, DNA), and in many applications, are preferablynon-biological polymers. In preferred embodiments, the polymer samplesare libraries of polymer samples, such as spatially separated librariesof polymer samples—for example, as a microtiter plate for analysis in ananalytical laboratory, or alternatively, such as temporally separatedsamples such as a series in time of on-line, near real time samples froman polymerization process line—for example, as part of a processmonitoring and/or process control system. The libraries of polymersamples can be provided on a common substrate. The libraries of polymersamples can be synthesized in parallel using, for example, a parallelpolymerization reactor. The libraries of polymer samples can comprisepolymer samples that are polymerization product mixtures resulting fromparallel polymerization reactions that are varied with respect to afactor affecting polymerization, such as one or more of reactantmaterials, catalysts, catalysts precursors, initiators, additives or therelative amounts thereof, or such as polymerization reaction conditions.The libraries of polymer samples can comprise polymer samples that areuntreated, or pretreated only with one or more steps selected from thegroup consisting of non-chromatographic separation, dilution, mixing andredissolution. Further detailed description about the nature of thepolymer samples, and/or of libraries of polymer samples, are included inthe Co-Owned Rapid Characterization of Polymers Application, a portionof which is reproduced as follows:

[0072] Polymer Samples

[0073] The present invention is particularly preferred in connectionwith the characterization of polymer samples, and especially,combinatorial libraries comprising different polymer samples. Thepolymer sample can be a homogeneous polymer sample or a heterogeneouspolymer sample, and in either case, comprises one or more polymercomponents. As used herein, the term “polymer component” refers to asample component that includes one or more polymer molecules. Thepolymer molecules in a particular polymer component have the same repeatunit, and can be structurally identical to each other or structurallydifferent from each other. For example, a polymer component may comprisea number of different molecules, with each molecule having the samerepeat unit, but with a number of molecules having different molecularweights from each other (e.g., due to a different degree ofpolymerization). As another example, a heterogeneous mixture ofcopolymer molecules may, in some cases, be included within a singlepolymer component (e.g., a copolymer with a regularly-occurring repeatunit), or may, in other cases, define two or more different polymercomponents (e.g., a copolymer with irregularly-occurring orrandomly-occurring repeat units). Hence, different polymer componentsinclude polymer molecules having different repeat units. It is possiblethat a particular polymer sample (e.g., a member of a library) will notcontain a particular polymer molecule or polymer component of interest.

[0074] The polymer molecule of the polymer component is preferably anon-biological polymer. A non-biological polymer is, for purposesherein, a polymer other than an amino-acid polymer (e.g., protein) or anucleic acid polymer (e.g., deoxyribonucleic acid (DNA)). Thenon-biological polymer molecule of the polymer component is, however,not generally critical; that is, the systems and methods disclosedherein will have broad application with respect to the type (e.g.,architecture, composition, synthesis method or mechanism) and/or nature(e.g., physical state, form, attributes) of the non-biological polymer.Hence, the polymer molecule can be, with respect to homopolymer orcopolymer architecture, a linear polymer, a branched polymer (e.g.,short-chain branched, long-chained branched, hyper-branched), across-linked polymer, a cyclic polymer or a dendritic polymer. Acopolymer molecule can be a random copolymer molecule, a block copolymermolecule (e.g., di-block, tri-block, multi-block, taper-block), a graftcopolymer molecule or a comb copolymer molecule. The particularcomposition of the non-biological polymer molecule is not critical, andcan include repeat units or random occurrences of one or more of thefollowing, without limitation: polyethylene, polypropylene, polystyrene,polyolefin, polyimide, polyisobutylene, polyacrylonitrile, poly(vinylchloride), poly(methyl methacrylate), poly(vinyl acetate),poly(vinylidene chloride), polytetrafluoroethylene, polyisoprene,polyacrylamide, polyacrylic acid, polyacrylate, poly(ethylene oxide),poly(ethyleneimine), polyamide, polyester, polyurethane, polysiloxane,polyether, polyphosphazine, polymethacrylate, and polyacetals.Polysaccharides are also preferably included within the scope ofnon-biological polymers. While some polysaccharides are of biologicalsignificance, many polysaccharides, and particularly semi-syntheticpolysaccharides have substantial industrial utility with little, if anybiological significance. Exemplary naturally-occurring polysaccharidesinclude cellulose, dextran, gums (e.g., guar gum, locust bean gum,tamarind xyloglucan, pullulan), and other naturally-occurring biomass.Exemplary semi-synthetic polysaccharides having industrial applicationsinclude cellulose diacetate, cellulose triacetate, acylated cellulose,carboxymethyl cellulose and hydroxypropyl cellulose. In any case, suchnaturally-occurring and semi-synthetic polysaccharides can be modifiedby reactions such as hydrolysis, esterification, alkylation, or by otherreactions.

[0075] In typical applications, a polymer sample is a heterogeneoussample comprising one or more polymer components, one or more monomercomponents and/or a continuous fluid phase. In copolymer applications,the polymer sample can comprise one or more copolymers, a firstcomonomer, a second comonomer, additional comonomers, and/or acontinuous fluid phase. The polymer samples can, in any case, alsoinclude other components, such as catalysts, catalyst precursors (e.g.,ligands, metal-precursors), solvents, initiators, additives, products ofundesired side-reactions (e.g., polymer gel, or undesired homopolymer orcopolymers) and/or impurities. Typical additives include, for example,surfactants, control agents, plasticizers, cosolvents and/oraccelerators, among others. The various components of the heterogeneouspolymer sample can be uniformly or non-uniformly dispersed in thecontinuous fluid phase.

[0076] The polymer sample is preferably a liquid polymer sample, such asa polymer solution, a polymer emulsion, a polymer dispersion or apolymer that is liquid in the pure state (i.e., a neat polymer). Apolymer solution comprises one or more polymer components dissolved in asolvent. The polymer solution can be of a, form that includeswell-dissolved chains and/or dissolved aggregated micelles. The solventcan vary, depending on the application, for example with respect topolarity, volatility, stability, and/or inertness or reactivity. Typicalsolvents include, for example, tetrahydro furan (THF), toluene, hexane,ethers, trichlorobenzene, dichlorobenzene, dimethylformamide, water,aqueous buffers, alcohols, etc. According to traditional chemistryconventions, a polymer emulsion can be considered to comprise one ormore liquid-phase polymer components emulsified (uniformly ornon-uniformly) in a liquid continuous phase, and a polymer dispersioncan be considered to comprise solid particles of one or more polymercomponents dispersed (uniformly or non-uniformly) in a liquid continuousphase. The polymer emulsion and the polymer dispersion can also beconsidered, however, to have the more typically employed meaningsspecific to the art of polymer science—of being aemulsion-polymerization product and dispersion-polymerization product,respectively. In such cases, for example, the emulsion polymer samplecan more generally include one or more polymer components that areinsoluble, but uniformly dispersed, in a continuous phase, with typicalemulsions including polymer component particles ranging in diameter fromabout 2 nm to about 500 nm, more typically from about 20 nm to about 400nm, and even more typically from about 40 nm to about 200 nm. Thedispersion polymer sample can, in such cases, generally include polymercomponent particles that are dispersed (uniformly or nonuniformly) in acontinuous phase, with typical particles having a diameter ranging fromabout 0.2 μm to about 1000 μm, more typically from about 0.4 μm to about500 μm, and even more typically from about 0.5 μm to about 200 μm.Exemplary polymers that can be in the form of neat polymer samplesinclude dendrimers, and siloxane, among others. The liquid polymersample can also be employed in the form of a slurry, a latex, a microgela physical gel, or in any other form sufficiently tractable for analysisas described and claimed herein. Liquid samples are useful in theautomated sample-handling tools that prepare and automatically sampleeach member of a polymer library. Liquid samples also allow the sampleto flow in the chromatographic system or characterization system. Insome cases, polymer synthesis reactions (i.e., polymerizations) directlyproduce liquid samples. These may be bulk liquid polymers, polymersolutions, or heterogeneous liquid samples such as polymer emulsions,lattices, or dispersions. In other cases, the polymer may besynthesized, stored or otherwise available for characterization in anon-liquid physical state, such as a solid state (e.g., crystalline,semicrystalline or amorphous), a glassy state or rubbery state. Hence,the polymer sample may need to be dissolved, dispersed or emulsified toform a liquid sample by addition of a continuous liquid-phase such as asolvent. The polymer sample can, regardless of its particular form, havevarious attributes, including variations with respect to polarity,solubility and/or miscibility.

[0077] In preferred applications, the polymer sample is a polymerizationproduct mixture. As used herein; the term “polymerization productmixture” refers to a mixture of sample components obtained as a productfrom a polymerization reaction. An exemplary polymerization productmixture can be a sample from a combinatorial library prepared bypolymerization reactions, or can be a polymer sample drawn off of anindustrial process line. In general, the polymer sample may be obtainedafter the synthesis reaction is stopped or completed or during thecourse of the polymerization reaction. Alternatively, samples of eachpolymerization reaction can be taken and placed into an intermediatearray of vessels at various times during the course of the synthesis,optionally with addition of more solvent or other reagents to arrest thesynthesis reaction or prepare the samples for analysis. Theseintermediate arrays can then be characterized at any time withoutinterrupting the synthesis reaction. It is also possible to use polymersamples or libraries of polymer samples that were prepared previouslyand stored. Typically, polymer libraries can be stored with agents toensure polymer integrity. Such storage agents include, for example,antioxidants or other agents effective for preventing cross-linking ofpolymer molecules during storage. Depending upon the polymerizationreaction, other processing steps may also be desired, all of which arepreferably automated. The polymerization scheme and/or mechanism bywhich the polymer molecules of the polymer component of the sample areprepared is not critical, and can include, for example, reactionsconsidered to be addition polymerization, condensation polymerization,step-growth polymerization, and/or chain-growth polymerizationreactions. Viewed from another aspect, the polymerization reaction canbe an emulsion polymerization or a dispersion polymerization reaction.Viewed more specifically with respect to the mechanism, thepolymerization reaction can be radical polymerization, ionicpolymerization (e.g., cationic polymerization, anionic polymerization),and/or ring-opening polymerization reactions, among others. Non-limitingexamples of the foregoing include, Ziegler-Natta or Kaminsky-Sinnreactions and various copolymerization reactions. Polymerization productmixtures can also be prepared by modification of a polymeric startingmaterials, by grafting reactions, chain extension, chain scission,functional group interconversion, or other reactions.

[0078] The sample size is not narrowly critical, and can generally vary,depending on the particular characterization protocols and systems usedto characterize the sample or components thereof. Typical sample sizescan range from about 0.1 μl to about 1 μl, more typically from about 1μl to about 1000 μl, even more typically from about 5 μl to about 100μl, and still more typically from about 10 μl to about 50 μl. Agenerally preferred sample size for flow characterization systems and,particularly for liquid chromatography, is a sample size of about 20 μl.

[0079] The polymer sample, such as a polymerization product mixture, canbe a raw, untreated polymer sample or can be pretreated in preparationfor characterization. Typical sample preparation steps includepreliminary, non-chromatographic separation of one or more components ofa polymer sample from other components, dilution, mixing and/orredissolution (e.g., from a solid state), among other operations.Preliminary separation methods can help remove large-scale impuritiessuch as dust, coagulum or other impurities. Such separation methods caninclude, for example: filtering (e.g., with a microfilter having poresizes that allow the passage of particles less than about 0.5 μm or 0.2μm); precipitation of polymer components, monomer components and/orother small-molecule components, decanting, washing, scavenging (e.g.,with drying agents), membrane separation (e.g., diafiltration,dialysis), evaporation of volatile components and/or ion-exchange. Thesample is preferably diluted, if necessary, to a concentration rangesuitable for detection. For typical liquid chromatography applications,for example, the sample concentration prior to loading into the liquidchromatography system can range from about 0.01 mg/ml to a neat sample,more typically from about 0.01 mg/ml to about 100 mg/ml, and even moretypically from about 0.1 mg/ml to about 50 mg/ml. More specificconcentration ranges typical for liquid chromatography samples includefrom about 0.1 mg/ml to about 20 mg/ml, and from about 0.5 mg/ml toabout 5 mg/ml. For flow-injection analysis systems, in which the sampleis detected without substantial chromatographic separation thereof, muchmore dilute solutions can be employed. Hence, the concentration canrange from a detectable concentration level (for the particular detectoremployed) up to about 1 mg/ml, or more in some applications. Typicalconcentrations can be about 1×10⁻² wt %, about 1×10⁻³ wt % or about1×10⁻⁴ wt %. Mixing can be required to increase the uniformity of apolymer sample emulsion or dispersion, and/or to integrate one or moreadditional components into the polymer sample. Preparation steps, andparticularly rapid preparation techniques, can be an important aspectfor combinatorial polymer investigations—since polymer samples may besynthesized in a form not ideally suited for immediate characterization.

[0080] Although the primary applications of the present invention aredirected to combinatorial polymer science research and/or qualitycontrol for industrial polymer synthesis or processing protocols,aspects of the invention can have applications involving non-polymersamples. A non-polymer sample can be a material that comprises anorganic or an inorganic non-polymer element or compound. Oligomers areconsidered to be polymers rather than non-polymers. The non-polymermolecule is, in some cases, preferably a non-biological non-polymerelement or compound. Such non-biological non-polymer elements orcompounds include non-polymer elements or compounds other than thosehaving a well-characterized biological activity and/or a primarycommercial application for a biological field (e.g., steroids, hormones,etc.). More particularly, such non-biological, non-polymer elements orcompounds can include organic or inorganic materials such as pigments,carbon powders (e.g., carbon black), metals, metal oxides, metal salts,metal colloids, metal ligands, etc, without particular limitation.

[0081] Pluralities of Samples/Libraries of Samples

[0082] A plurality of samples such as polymer samples comprises 2 ormore samples that are physically or temporally separated from eachother—for example, by residing in different sample containers, by havinga membrane or other partitioning material positioned between samples, bybeing partitioned (e.g., in-line) with an intervening fluid, by beingtemporally separated in a flow process line (e.g., as sampled forprocess control purposes), or otherwise. The plurality of samplespreferably comprises 4 or more samples, more preferably 8 or moresamples, and even more preferably 10 or more samples. Four samples canbe employed, for example, in connection with experiments having onecontrol sample and three polymer samples varying (e.g., with respect tocomposition or process conditions as discussed above) to berepresentative of a high, a medium and a low-value of the variedfactor—and thereby, to provide some indication as to trends. Eightsamples can provide for additional variations in the explored factorspace. Moreover, eight samples corresponds to the number of parallelpolymerization reactors in the PPR-8™, being selectively offered as oneof the Discovery Tools™ of Symyx Technologies, Inc. (Santa Clara,Calif.). Higher numbers of samples can be investigated, according to themethods of the invention, to provide additional insights into largercompositional and/or process space. In some cases, for example, theplurality of samples can be 15 or more samples, preferably 20 or moresamples, more preferably 40 or more samples and even more preferably 80or more samples. Such numbers can be loosely associated with standardconfigurations of parallel reactor configurations (e.g., the PPR-48™,Symyx Technologies, Inc.) and/or of standard sample containers (e.g.,96-well microtiter plate-type formats). Moreover, even larger numbers ofsamples such as polymer samples can be characterized according to themethods of the present invention for larger scale research endeavors.Hence, the number of samples can be 150 or more, 400 or more, 500 ormore, 750 or more, 1,000 or more, 1,500 or more, 2,000 or more, 5,000 ormore and 10,000 or more polymer samples. As such, the number of samplescan range from about 2 samples to about 10,000 samples, and preferablyfrom about 8 samples to about 10,000 samples. In many applications,however, the number of samples can range from about 80 samples to about1500 samples. In some cases, in which processing of samples usingtypical 96-well microtiter-plate formatting is convenient or otherwisedesirable, the number of samples can be 96*N, where N is an integerranging from about 1 to about 100. For many applications, N can suitablyrange from 1 to about 20, and in some cases, from 1 to about 5.

[0083] The plurality of samples can be a combinatorial library ofsamples. A library of samples comprises of two or more differentsamples, and can be in an array format as spatially separatedsamples—preferably on a common substrate, or temporally separated—forexample, in a flow system. Candidate samples (i.e., members) within alibrary may differ in a definable and typically predefined way,including with regard to chemical structure, processing (e.g.,synthesis) history, mixtures of interacting components, purity, etc. Thesamples can be spatially separated, preferably at an exposed surface ofthe substrate, such that the array of samples are separately addressablefor sampling into the characterization system and subsequentcharacterization thereof. The two or more different samples can residein sample containers formed as wells in a surface of the substrate. Thenumber of samples included within the library can generally be the sameas the number of samples included within the plurality of samples, asdiscussed above. In general, however, not all of the samples within alibrary of samples need to be different samples. When process conditionsare to be evaluated, the libraries may contain only one type of sample.Typically, however, for combinatorial polymer science researchapplications, at least two or more, preferably at least four or more,even more preferably eight or more and, in many cases most, andallowably each of the plurality of polymer samples in a given library ofpolymer samples will be different from each other. Specifically, adifferent polymer sample can be included within at least about 50%,preferably at least 75%, preferably at least 80%, even more preferablyat least 90%, still more preferably at least 95%, yet more preferably atleast 98% and most preferably at least 99% of the polymer samplesincluded in the sample library. In some cases, all of the polymersamples in a library of polymer samples will be different from eachother.

[0084] The substrate can be a structure having a rigid or semi-rigidsurface on which or into which the array of polymer samples can beformed or deposited. The substrate can be of any suitable material, andpreferably consists essentially of materials that are inert with respectto the polymer samples of interest. Certain materials will, therefore,be less desirably employed as a substrate material for certainpolymerization reaction process conditions (e.g., hightemperatures—especially temperatures greater than about 100 C—or highpressures) and/or for certain reaction mechanisms. Stainless steel,silicon, including polycrystalline silicon, single-crystal silicon,sputtered silicon, and silica (SiO₂) in any of its forms (quartz, glass,etc.) are preferred substrate materials. Other known materials (e.g.,silicon nitride, silicon carbide, metal oxides (e.g., alumina), mixedmetal oxides, metal halides (e.g., magnesium chloride), minerals,zeolites, and ceramics) may also be suitable for a substrate material insome applications. Organic and inorganic polymers may also be suitablyemployed in some applications of the invention. Exemplary polymericmaterials that can be suitable as a substrate material in particularapplications include polyimides such as Kapton™, polypropylene,polytetrafluoroethylene (PTFE) and/or polyether etherketone (PEEK),among others. The substrate material is also preferably selected forsuitability in connection with known fabrication techniques. As to form,the sample containers formed in, at or on a substrate can be preferably,but are not necessarily, arranged in a substantially flat, substantiallyplanar surface of the substrate. The sample containers can be formed ina surface of the substrate as dimples, wells, raised regions, trenches,or the like. Non-conventional substrate-based sample containers, such asrelatively flat surfaces having surface-modified regions (e.g.,selectively wettable regions) can also be employed. The overall sizeand/or shape of the substrate is not limiting to the invention. The sizeand shape can be chosen, however, to be compatible with commercialavailability, existing fabrication techniques, and/or with known orlater-developed automation techniques, including automated sampling andautomated substrate-handling devices. The substrate is also preferablysized to be portable by humans. The substrate can be thermallyinsulated, particularly for high-temperature and/or low-temperatureapplications. In preferred embodiments, the substrate is designed suchthat the individually addressable regions of the substrate can act aspolymerization reaction vessels for preparing a polymerization productmixture (as well as sample containers for the two or more differentpolymer samples during subsequent characterization thereof. Glass-lined,96-well, 384-well and 1536-well microtiter-type plates, fabricated fromstainless steel and/or aluminum, are preferred substrates for a libraryof polymer samples. The choice of an appropriate specific substratematerial and/or form for certain applications will be apparent to thoseof skill in the art in view of the guidance provided herein.

[0085] The library of polymer materials can be a combinatorial libraryof reaction product mixtures such as polymerization product mixtures.Polymer libraries can comprise, for example, polymerization productmixtures resulting from polymerization reactions that are varied withrespect to, for example, reactant materials (e.g., monomers,comonomers), catalysts, catalyst precursors, initiators, additives, therelative amounts of such components, reaction conditions (e.g.,temperature, pressure, reaction time) or any other factor affectingpolymerization. Design variables for polymerization reactions are wellknown in the art. See generally, Odian, Principles of Polymerization,3^(rd) Ed., John Wiley & Sons, Inc. (1991). A library of polymer samplesmay be prepared in arrays, in parallel polymerization reactors or in aserial fashion. Exemplary methods and apparatus for preparing polymerlibraries—based on combinatorial polymer synthesis approaches—aredisclosed in copending U.S. patent application Ser. No. 09/211,982 ofTurner et al. filed Dec. 14, 1998, copending U.S. patent applicationSer. No. 09/227,558 of Turner et al. filed Jan. 8, 1999, copending U.S.patent application Ser. No. 09/235,368 of Weinberg et al. filed Jan. 21,1999, and copending U.S. provisional patent application Ser. No.60/122,704 entitled “Controlled, Stable Free Radical Emulsion andWater-Based Polymerizations”, filed Mar. 9, 1999 by Klaerner et al.under Attorney Docket No. 99-4. See also, PCT Patent Application WO96/11878.

[0086] The libraries can be advantageously characterized directly,without being isolated, from the reaction vessel in which the polymerwas synthesized. Thus, reagents, catalysts or initiators and otheradditives for making polymers may be included with the polymer samplefor characterization or screening.

[0087] While such methods are preferred for a combinatorial approach topolymer science research, they are to be considered exemplary andnon-limiting. As noted above, the particular polymer samplescharacterized according to the methods and with the apparatus disclosedherein can be from any source, including, but not limited topolymerization product mixtures resulting from combinatorially synthesisapproaches.

[0088] Mini- and Micro-Scale Applications

[0089] The methods of the present invention can be applied in connectionwith “normal” scale HPLC systems, and can also be applied to smallerscale systems—including particularly mini-scale systems and micro-scalesystems. As used herein, mini-scale systems are considered to includethose having mobile-phase supply conduits and/or separation units (e.g.,chromatographic columns) with a diameter ranging from about 3 mm toabout 500 μm, and micro-scale systems are considered to include thosehaving mobile-phase supply conduits with a diameter of about 500 μm orless. For other than circular cross-sections, equivalent dimensions canbe determined based on hydraulic radius.

[0090] Preferably at least one of the first or second dimensions arehigh-performance liquid chromatography subsystems adapted for gelpermeation chromatography. In a particularly preferred approach, thefirst dimension HPLC subsystem can be adapted for chromatographicapproaches effective for distinguishing between chemical compositionand/or structural variations of polymer sample components (e.g., repeatunits types, ratios of copolymer repeat units, functional groups,branching, etc.). Exemplary preferred first-dimension HPLC subsystemsinclude reverse phase chromatography subsystems, mobile-phasecompositional gradient elution chromatography subsystems (e.g.,compositionally-varying mobile phase gradients and/ortemperature-varying mobile phase gradients), or mobile-phase temperaturegradient elution chromatography subsystems. Mobile-phase elutiongradients of the first dimension preferably comprise a substantiallyuniversal co-solvent system, such as a water-tetrahydrofuran-hexanesystem. Generally, in this particularly preferred approach, the seconddimension HPLC subsystem is preferably adapted for size-exclusionchromatography (SEC) such as gel permeation chromatography (GPC).

[0091] The methods can further comprise determining a property ofinterest from the detected property of the first-dimension and/or seconddimension. The detector type is not generally critical, and can includefor example, mass detectors and/or concentration detectors. Evaporativelight scattering detectors (ELSD) are preferred in some embodiments.Further details about detection, including types of detectors andvarious combinations of detectors, and including various types ofdetected and/or determined properties, is described in detail in theattached Co-Owned Rapid Characterization of Polymers Application.

[0092] The multi-dimensional HPLC system of the invention is preferablyoperated under the control of one or more microprocessors (not shown),preferably configured with software effective for operating the hardware(sampling systems, injection valves, mobile-phase pumps, detectionsystems) and for effecting tracking and acquiring data, etc. Suchsuitable software is commercially available, for example, from liquidchromatography systems manufacturers, such as Millenium software(Waters), and/or from software manufacturers, such as Lab View brandsoftware. The software can, if necessary, be modified to incorporatefunctionality for driving the aforementioned hardware and data trackingand acquisition needs for the first and second dimension HPLCsubsystems. In a preferred embodiment, for example, with reference toFIG. 1, Lab View software can be modified to (i) integrate withImpressionist™ robotic-control software (Symyx Technologies, Inc., SantaClara, Calif.) used for controlling the robotic pipette (CavroInstruments, Inc.) hardware for serially withdrawing polymer samples 100from a library of polymer samples, and for injecting such polymersamples into a loading port 1310 of a first-dimension injector 1300,such integration including tracking of timing of injection as aninitiation point for the two-dimensional chromatography analysismethodologies programmed into the Lab View software; (ii) to controlfirst-dimension HPLC analysis operations, including first-dimensionmobile phase pumps 1200 to control first-dimension mobile phase flowrates, and if desired, temperature control of the mobile phase and/orcolumn, and if desired, first-dimension mobile phase source selectionvalves (not shown) for providing mobile phase gradients forfirst-dimension gradient elution chromatography; (iii) to controlsecond-dimension HPLC analysis operations, including thesecond-dimension injector 2300 for comprehensive, directly coupledsampling from the first dimension to the second dimension (e.g., such asregularly recurring interval sampling), the mobile phase pumps 2200 tocontrol second-dimension mobile phase flow rates, and if desired,temperature control of the mobile phase and/or column, and if desired,second-dimension mobile phase source selection valves (not shown) forproviding mobile phase gradients for first-dimension gradient elutionchromatography, the second-dimension detector(s) 2600 for dataacquisition and handling, etc.

[0093] As shown in FIGS. 3A and 3B, for example, the customized Lab Viewsoftware can include a graphical user interface that allows forefficient user-driven control of such hardware and data managementfunctions, as well as for integrated or separate display of resultingcharacterization data. Briefly, FIG. 3A shows a graphical user interfacecomputer screen shot 3000 that includes a set-up and control panel 3100and a data readout panel 3300. The set-up and control panel 3100comprises several subpanels or sections, including a gradient zonesselection section 3110, a 2-D injection zone selection section 3120, aprogrammed gradient profile display section 3130, a pre-flow indicatordisplay section 3140, a pre-injection selection section 3150, a commentsection 3160, and an indicator section 3170. The pre-injection selectionsection 3150 comprises data entry boxes for selecting pre-injection flowrates, pre-injection fraction of one of the mobile-phase souces, andpre-injection fraction of another of the mobile-phase sources. Thegradient zone selection section 3110 includes drop-down data entry boxesfor selecting gradient zones, zone start times, total flow rates,fraction of one solvent of the mobile-phase gradient, and fraction ofanother solvent of the mobile phase gradient. The 2-D injection zoneselection section 3120 comprises drop-down data entry boxes for definingthe time of the gradient zones, a selection tab for optionally selectingevenly spaced gradient zones, and a drop-down data entry box fordefining the duration of the second-dimension analysis. The programmedgradient profile display section 3130 comprises a graphical display areafor showing the programmed gradient profile for each of the mobile-phasesources (e.g., solvents). The pre-flow indicator display section 3140comprises a graphical display area for graphically displaying thepre-injection flow rate over time. The comment section 3160 includes adata entry box for entering comments. The indicator section 3170includes a red indicator light for indicating waiting for firstinjection, and a second green indicator light for indicating that thesystem is recording data. The data readout panel 3300 also comprisesseveral subpanels or sections, including a pump flow rate indication anddisplay section 3310, a pump-pressure indication and display section3320, a pump zone indication section 3330, a 2-D zone indication section3340, a UV detector output display section 3350, an ELSD output displaysection 3360, a UV output from 2^(nd) injection display section 3370,and a final output display section 3380. The pump flow rate indicationand display section 3310 comprises indicator boxes and a display panelfor pump flow rates, together with a display panel for a legend of thedisplay panel, the pump-pressure indication and display section 3320comprises indicator boxes and a display panel for pump pressure,together with a display panel for a legend of the display panel, thepump zone indication section 3330 comprises an indicator box for pumpzones, the 2-D zone indication section 3340 comprises an indicator boxfor 2-dimensional zone, the UV detector output display section 3350comprises a display box for the UV detector data, the ELSD outputdisplay section 3360 comprises a display box for the ELSD detector data,the UV output from 2^(nd) injection display section 3370 comprises adisplay box for all of the 2^(nd) injection UV detector data, togetherwith a display panel for the legend thereof, and a final output displaysection 3380 comprising a display box for the final output data,together with a display panel for a legend thereof. FIG. 3B shows agraphical user interface computer screen shot 4000 that includes a3-dimensional graphical display panel 4100 and a two-dimensionalgraphical display panel 4200, as well as a raw data display panel 4300.The 3-dimensional graphical display panel 4100 comprises athree-dimensional representation of the detector response versusfirst-dimension retention time and versus second-dimension retentiontime, together with a legend display panel. The two-dimensionalgraphical display panel 4200 comprises a two-dimensional contour plot offirst-dimension retention time versus second-dimension retention time,as well as a legend display panel. The raw data display panel 4300comprises a table of raw data.

[0094] Further details about microprocessor control of the HPLCsubsystems, is described in detail in the attached Co-Owned RapidCharacterization of Polymers Application and in the attached Co-OwnedParallel HPLC Application.

[0095] The following examples illustrate the principles and advantagesof the invention.

EXAMPLES Example 1

[0096] Two-Dimensional Liquid Chromatography with Regularly Recurring2^(nd)-Dimension Sampling Interval

[0097] This example demonstrates two-dimensional liquid chromatographytechniques for characterizing a polymer sample comprising polymercomponents of varying composition and/or molecular weight, wherein thesecond-dimension is comprehensively and directly coupled to the firstdimension, by in-line sampling of the first-dimension mobile phaseeluent at regularly recurring intervals of time.

[0098] In experiments effected for this example, various polymer samples(described below) having components with different chemical compositionsand/or molecular weight were characterized in a two-dimensionchromatography system comprising a first dimension HPLC subsystemadapted for normal-phase compositional gradient elution chromatography,and a second dimension HPLC subsystem adapted for gel permeationchromatography (GPC). Briefly, about 50 μL of a polymer sample solutionwas injected into the mobile phase of the first dimension, andchromatographically separated in a first-dimension HPLC column havingseparation media effective for normal-phase separation, using a relayhexane-tetrahydrofuran water gradient elution at a first-dimension flowrate of about 0.5 mL/min. The first-dimension mobile phase eluent comingof the first-dimension HPLC column was sampled using a multiportinjection valve configured with two sample loops, each having a volumeof about 250 μl. Discrete volume fractions (250 μl) of thefirst-dimension mobile phase eluent were sampled at regularly recurringintervals with a frequency of about 30 seconds per sample, and thesampled portions were injected at the same time frequency into asecond-dimension mobile phase. Chromatographic separation in thesecond-dimension was effected using a second-dimension column havingseparation media effective for size-exclusion chromatography (SEC),specifically, gel permeation chromatography (GPC), and using DMF as themobile phase at a second-dimension flow rate of about 4 mL/min.Detection was effected by routing all of the eluent from the GPC columninto an evaporative light scattering detector, the signal of which isproportional to the concentration of polymer in the second-dimensionmobile phase eluent. A 2-dimensional (2-D) chromatogram or 2-D map wascomposed by stacking individual chromatograms corresponding to 30 secondintervals that represent the SEC chromatographic traces. The firstdimension retention time was represented by the time of sampling of aparticular fraction from the first-dimension mobile phase eluent forinjection into the second-dimension.

[0099] In a first experiment, the second-dimension SEC subsystem wascalibrated by characterizing a set of polystyrene polymer standardscombined as a single sample—as a single “shot” in the aforementionedtwo-dimensional liquid chromatography system. Hence, the polymer samplecomprised a set of polystyrene polymer standards as polymer componentshaving the same chemical composition, but different known molecularweights. The results, shown in FIGS. 4A and 4B, demonstrate that thesecond dimension provides adequate resolution of polymer samplecomponents having different molecular weights in less than about 30seconds. Specifically, FIG. 4A is a graph showing detector response (mV)versus retention time (min) with clear resolution of polystyrenestandards subcomponents of different molecular weights. FIG. 4B is agraph showing the corresponding log molecular weigh data versus-retention time (min) with the expected substantially linearrelationship between components of the polystyrene standards sample.Hence, this first experiment demonstrates that adequate molecular weightresolution can be achieved in the second-dimension HPLC (SEC/GPC)subsystem with a regularly-recurring sampling interval from the firstdimension, and injection to injection interval into the second dimensionof about 30 seconds.

[0100] In a second experiment, a polymer solution comprising differenttypes of polymers as sample polymer components was characterized todetermine chemical composition distribution and molecular weightdistribution in a single-shot sample analysis with a run time of about15 minutes. Specifically, the polymer sample comprisedpolyhydroxyethylmethacrylate (PHEMA), polymethylmethacrylate (PMMA) andpolystyrene (PS) components, each component having roughly the samemolecular weight distribution. This polymer sample was characterized inthe two-dimension chromatography system described above (normal phaseHPLC first dimension/SEC (GPC) second dimension) using substantially thesame operational protocols as described. The results, shown in FIGS. 5Aand 5B, demonstrate that the first dimension provides adequateresolution of polymer sample components having different chemicalcompositions, but substantially the same molecular weight distributionunder the aforementioned conditions and separation protocols, with abouta 15 minute total run time (about 14½ minutes of which were for thefirst-dimension characterization, and about ½ minute of which was forthe second dimension characterization). Specifically, FIG. 5A is a3-dimensional plot showing detector response (V) versus both (i)normal-phase HPLC retention time (min), corresponding to thefirst-dimension separation, and (ii) GPC retention time (min),corresponding to the second-dimension separation, with clear resolutionof the various types of polymer components in the polymer sample. FIG.5B is a 2-dimensional contour graph showing the corresponding top-downview of the data presented in FIG. 5A, including normal-phase HPLCretention time (min), corresponding to the first-dimension separationversus GPC retention time (min), corresponding to the second-dimensionseparation, again showing clear resolution of the various types ofpolymer components in the polymer sample.

[0101] In a third experiment, a polymer solution comprising samplecomponents that included different types of polymers with differentmolecular weights, as well as the same types of polymers with differentmolecular weights, was characterized to determine chemical compositiondistribution and molecular weight distribution in a single-shot sampleanalysis. Specifically, the polymer sample comprisedpolymethylmethacrylate (PMMA, ˜100 K molecular weight),polyethyleneoxide (PEO, ˜1.7 M molecular weight), polyethyleneoxide(PEO, ˜3 K molecular weight), polystyrene (PS, ˜3 M molecular weight),and polystyrene (PS, ˜10 K molecular weight) as components thereof. Thispolymer sample was characterized in the two-dimension chromatographysystem described above (normal phase HPLC first dimension/SEC (GPC)second dimension) using substantially the same operational protocols asdescribed. The results, shown in FIG. 6, demonstrate that thetwo-dimensional liquid chromatography system provides adequateresolution of these polymer sample components—both with respect tochemical composition distribution and molecular weight distributionunder the aforementioned conditions and separation protocols.Specifically, FIG. 6 is a 2-dimensional graph showing the relativenormal-phase HPLC retention time, corresponding to the first-dimensionseparation, versus the relative GPC retention time (min), correspondingto the second-dimension separation. Clear resolution of the varioustypes of polymer components in the polymer sample is demonstrated.

Example 2

[0102] Use of 2-Dimensional Liquid Chromatography for FingerprintingCharacterization of a Combinatorial Library of Polymer Samples

[0103] This example demonstrates two-dimensional liquid chromatographytechniques as applied for characterizing a combinatorial library ofpolymer samples comprising polymer components of random copolymers,poly(AB), synthesized by free-radical polymerization in a parallel batchreactor with various ratios of monomers A and B, and with varying ratiosof monomer to initiator. This example also demonstrates that suchtwo-dimensional characterization protocols are comparable in quality ofresults to separate, one-dimensional analysis conducted independently ofeach other, and favorable in terms of overall sample throughput.

[0104] The polymer samples of the library were synthesized in theparallel batch reactor using combinatorial polymerization approachesknown in the art. FIG. 7A is a graphical representation of the librarydesign for the library of polymer samples, showing that (i) the relativeratio of monomer A to monomer B ranges from about 0 to about 1 alongeach of the rows of the synthesis reactor (microtiter-type format), andis about the same in each of the columns thereof, and that (ii) therelative ratio of monomer to initiator increases along each of thecolumns of the synthesis reactor (moving from top to bottom, as shown),and is about the same in each of the rows thereof.

[0105] The polymer samples of the library were characterized in acomprehensive, directly-coupled two-dimension liquid chromatographysystem comprising a first dimension HPLC subsystem adapted fornormal-phase compositional gradient elution chromatography, and a seconddimension HPLC subsystem adapted for gel permeation chromatography(GPC). The operational conditions and protocols for the two-dimensionalliquid chromatography system was substantially the same as thatdescribed in connection with Example 1. The results, shown in FIGS. 7Band 7C, demonstrate that the two-dimension HPLC system providessubstantial resolution of polymer sample fingerprints—chemicalcomposition and molecular weight data for each of the polymer samples ofthe library. Specifically, FIG. 7B is a 3-dimensional plot showingmolecular weight, as determined from second-dimension GPC data versuspolystyrene standard calibration, versus spatial position in themicrotiter-format parallel reactor (columns 1-12 and rows 1-7). FIG. 7Cis a 3-dimensional plot showing chemical composition, as determined fromfirst-dimension normal phase HPLC data (and shown as % of monomer Bincorporated into each of the random copolymer samples), versus spatialposition in the microtiter-format parallel reactor (columns 1-12 androws 1-7).

Example 3

[0106] Comparative Characterization Using 2-Dimensional LiquidChromatography versus Using Separate, One-Dimension HPLC and GPCAnalysis, for Fingerprinting Characterization of a Combinatorial Libraryof Polymer Samples

[0107] This example demonstrates a comparison between two-dimensionalliquid chromatography techniques, and corresponding separate, individualone-dimensional liquid chromatography protocols, as applied forcharacterizing a combinatorial library of polymer samples. The libraryof polymer samples comprises polymer components of random copolymers,poly(AB), synthesized by free-radical polymerization in a parallel batchreactor with various ratios of monomers A and B, and with varying ratiosof monomer to initiator, where A and B representhydroxyethylmethacrylate (HEMA) and styrene monomers, respectively.

[0108] The polymer samples of the library were synthesized in theparallel batch reactor using combinatorial polymerization approachesknown in the art. FIG. 8A is a graphical representation of the librarydesign for the library of polymer samples, showing that (i) the relativeratio of monomer A to monomer B ranges from about 0 to about 1 alongeach of the rows of the synthesis reactor (microtiter-type format), andis about the same in each of the columns thereof, and that (ii) therelative ratio of monomer to initiator increases along each of thecolumns of the synthesis reactor (moving from top to bottom, as shown),and is about the same in each of the rows thereof.

[0109] The polymer samples of the library were characterized in acomprehensive, directly-coupled two-dimension liquid chromatographysystem comprising a first dimension HPLC subsystem adapted fornormal-phase compositional gradient elution chromatography, and a seconddimension HPLC subsystem adapted for gel permeation chromatography(GPC). The operational conditions and protocols for the two-dimensionalliquid chromatography system was substantially the same as thatdescribed in connection with Example 1. The results of thetwo-dimensional characterization are shown in FIG. 8B. FIG. 8B is anarray of 2-dimensional contour graphs, each graph representing data fromone of the samples of the library, and each graph showing chemicalcomposition distribution (represented as normal phase HPLC retentiontime, corresponding to the relative amount of monomer B in each of thesamples), versus molecular weight distribution (represented as GPCretention time (-log MW)). Note that for simplicity of presentation, thedata included in FIG. 8B corresponds to only three rows of the polymersamples of the library of FIG. 8A.

[0110] For comparative purposes, the library of polymer samples (FIG.8A) was characterized using two separate HPLC characterizationtechniques, as one-dimensional analysis independent of each other:normal phase HPLC gradient elution chromatography and rapid-analysis GPCchromatography, each of which was effected using conditions andprotocols substantially the same as those used in the first-dimensionsubsystem and second-dimensions subsystem of the two-dimensionalanalysis (see Example 1). FIG. 8C shows the results of the independent,one-dimensional analysis for the same polymer samples for which data isshown in FIG. 8B. Specifically, FIG. 8C is an array of 2-dimensionalplots, each plot representing the combined, independently-obtained datafrom one of the samples of the library, and each plot showing chemicalcomposition (represented as the relative amount of monomer B in each ofthe samples as determined by the independent, one-dimension normal phaseHPLC gradient elution characterization), versus molecular weight(represented as GPC log MW). Note that the molecular weight units inFIG. 8C are the opposite in sign from those of FIG. 8B, requiring aninversion of data for comparison purposes between FIGS. 8B and 8C.

[0111] Comparison of FIGS. 8B and 8C demonstrates that thetwo-dimensional characterization protocols are comparable or favorableto separate, one-dimensional analysis conducted independently of eachother, with respect to quality of analysis results and the capability toidentify trends and associate those trends with the polymerizationsynthesis conditions. Significantly, the data obtained from thetwo-dimensional characterization scheme allows for distribution dataprofiles, whereas the two separate, independent one-dimensional analysisallows for only overall molecular weight/chemical compositioninformation. Additionally, the two-dimensional chromatography wasfavorable to the separate, one-dimensional analysis approach in terms ofoverall sample throughput.

Example 4

[0112] Comparative Characterization Using 2-Dimensional LiquidChromatography Versus Using Traditional GPC-FTIR Analysis, for PolymerSample Fingerprinting

[0113] This example demonstrates a comparison between two-dimensionalliquid chromatography techniques, and traditional gel permeationchromatography-Fourier transformed infrared (GPC-FTIR) analysis forfingerprinting of a polymer sample. The example shows, in particular,that two-dimensional liquid chromatography is a more definitivetechnique for distinguishing whether the polymer sample comprises apolymer blend of two different polymer types and/or molecular weights,or whether the polymer sample comprises a single copolymer. Moreover,the more definitive result of the two-dimensional analysis was obtainedin about 15 minutes—about ⅛ of the overall characterization time usingthe traditional GPC-FTIR approach (about 2 hours).

[0114] The polymer comprised polymethylmethacrylate (PMMA) andpolystyrene (PS) components, each component having roughly the samemolecular weight distribution.

[0115] This polymer sample was first characterized in the two-dimensionchromatography system described in connection with Example 1 (normalphase HPLC first dimension/SEC (GPC) second dimension) usingsubstantially the same operational protocols as described in Example 1,with a total sample analysis time of about 15 minutes. The results,shown in FIG. 9A, demonstrate that the two-dimensional characterizationapproach resolved the sample components into separate components,thereby allowing for positive identification of the polymer sample as ablend of the two polymer components. Specifically, FIG. 9A is a2-dimensional contour graph showing normal-phase HPLC retention time(sec), corresponding to the first-dimension separation, versus GPCretention time (min), corresponding to the second-dimension separation,with clear resolution of the polymer components.

[0116] This sample was also characterized in a traditional GPC-FTIRanalysis system, in which fractions from a GPC system were spotted ontoan array, and subsequently analyzed using FTIR techniques. The totalanalysis time required about two hours. The results, shown in FIG. 9B,demonstrate that the analysis remains inconclusive as to whether thepolymer sample was a copolymer or whether it comprised a blend of twopolymer components.

Example 5

[0117] Use of 2-Dimensional Liquid Chromatography for Tuning ofPolymerization Synthesis Conditions During Scale Up of PolymerizationProcess

[0118] This example demonstrates two-dimensional liquid chromatographytechniques for fingerprinting characterization as applied for tuning ofpolymerization synthesis conditions in an effort to scale up apolymerization process (e.g., from bench scale to pilot scale or frompilot scale to commercial production scale). The example alsodemonstrates two-dimensional liquid chromatography techniques as appliedfor polymerization process monitoring and/or polymerization processcontrol

[0119] In a first experiment, a library of polymer samples weresynthesized and screened using combinatorial (high-throughput)techniques known in the art. One of the polymer samples was determinedto have useful properties in the library-scale. The polymer sample ofinterest was a random copolymer. The polymer sample of interest of thelibrary was characterized in a comprehensive, directly-coupledtwo-dimension liquid chromatography system comprising a first dimensionHPLC subsystem adapted for normal-phase compositional gradient elutionchromatography, and a second dimension HPLC subsystem adapted for gelpermeation chromatography (GPC). The operational conditions andprotocols for the two-dimensional liquid chromatography system wassubstantially the same as that described in connection with Example 1.The results are shown in FIG. 10, as a 2-dimensional contour graphshowing chemical composition distribution, represented by polarity (asdetermined using normal-phase HPLC retention time in a first-dimensionanalysis), versus molecular weight distribution (as determined using GPCretention time in a second-dimension analysis).

[0120] During a scale up effort to prepare the same polymer sample ofinterest on a larger scale (scaling factor of about 1:1000), thetwo-dimensional chromatography analysis (under the same operationalprotocols and conditions) was used to tune the polymerization synthesisconditions for the scaled-up process, until the results of thetwo-dimensional analysis for the large-scale synthesis polymer sample,shown in FIG. 10B, was substantially the same as the results for thesmall scale synthesis polymer sample (FIG. 10A). As shown, FIG. 2B showsa 2-dimensional contour graph showing chemical composition distribution,represented by polarity (as determined using normal-phase HPLC retentiontime in a first-dimension analysis), versus molecular weightdistribution (as determined using GPC retention time in asecond-dimension analysis) for the large-scale synthesis polymer sample.

[0121] In light of the detailed description of the invention and theexamples presented above, it can be appreciated that the several objectsof the invention are achieved.

[0122] The explanations and illustrations presented herein are intendedto acquaint others skilled in the art with the invention, itsprinciples, and its practical application. Those skilled in the art mayadapt and apply the invention in its numerous forms, as may be bestsuited to the requirements of a particular use. Accordingly, thespecific embodiments of the present invention as set forth are notintended as being exhaustive or limiting of the invention.

1. A method for characterizing a polymer sample in a multi-dimensionalliquid chromatography system, the method comprising: injecting thepolymer sample into a first-dimension mobile phase of a first dimensionof the multi-dimensional liquid chromatography system;chromatographically separating at least one sample component of theinjected polymer sample from other sample components thereof in afirst-dimension liquid chromatography column, such that afirst-dimension mobile phase eluent from the first-dimension columncomprises two or more first-dimension separated sample components;sampling discrete volumes of the first-dimension mobile phase eluent atregularly recurring time intervals, such that at least a portion of eachof the first-dimension separated sample components are sampled;injecting each of the sampled volumes of the first-dimension mobilephase eluent directly into a second-dimension mobile phase of the seconddimension of the multi-dimensional liquid chromatography system;chromatographically separating at least one subcomponent of the sampledportions of each of the first-dimension separated sample components fromother subcomponents thereof in a second-dimension liquid chromatographycolumn, such that a second-dimension mobile phase eluent from thesecond-dimension column comprises two or more second-dimension separatedsubcomponents for each of the sampled portions of each of thefirst-dimension separated sample components; and detecting a property ofthe second-dimension separated subcomponents in the second-dimensionmobile phase eluent using a flow-through detector.
 2. The method ofclaim 1, further comprising controlling the sampling interval andsampling volume such that at least two discrete fractions of each of thefirst-dimension separated sample components are sampled.
 3. The methodof claim 1, further comprising controlling the sampling interval andsampling volume such that at least three discrete fractions of each ofthe first-dimension separated sample components are sampled.
 4. Themethod of claim 1, wherein a discrete volume of the first-dimensionmobile phase eluent is sampled at least once every two minutes.
 5. Themethod of claim 1, wherein a discrete volume of the first-dimensionmobile phase eluent is sampled at least once every 30 seconds.
 6. Themethod of claim 1, wherein at least ten discrete volumes of thefirst-dimension mobile phase eluent are sampled.
 7. The method of claim1, wherein at least one hundred discrete volumes of the first-dimensionmobile phase eluent are sampled.
 8. The method of claim 1, wherein thesampled volumes of the first-dimension mobile phase eluent are not morethan about 500 μl.
 9. The method of claim 1, wherein the sampled volumesof the first-dimension mobile phase eluent are not more than about 250μl.
 10. The method of claim 1, wherein at least ten discrete volumes ofthe first-dimension mobile phase eluent are sampled with a samplingfrequency of at least once every 30 seconds, and the sampled volumes arenot more than about 250 μl.
 11. The method of claim 1, wherein at leastone of the sampled volumes of the first-dimension mobile phase eluenthas an essential absence of first-dimension separated sample components.12. The method of claim 1, further comprising determining a property ofinterest from the detected property of the second-dimension separatedsubcomponents.
 13. The method of claim 1, further comprising detecting aproperty of the first-dimension separated components in thefirst-dimension mobile phase eluent using a flow-through detector. 14.The method of claim 1, wherein the property of the second-dimensionseparated subcomponents is detected using a concentration detector ormass detector.
 15. The method of claim 1, wherein the property of thesecond-dimension separated subcomponents is detected using anevaporative light-scattering detector.
 16. The method of claim 1,wherein at least one of the first dimension or second dimension of themulti-dimensional liquid chromatography system is a high-performanceliquid chromatography subsystem adapted for size exclusionchromatography.
 17. The method of claim 1, wherein at least one of thefirst dimension or second dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for gel permeation chromatography.
 18. The method ofclaim 1, wherein the second dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for size exclusion chromatography.
 19. The method ofclaim 1, wherein the second dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for gel permeation chromatography.
 20. The method ofclaim 1, wherein the first dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for determining compositional variations offirst-dimension separated sample components.
 21. The method of claim 1,wherein the first dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for mobile-phase compositional gradient elutionchromatography.
 22. The method of claim 1, wherein the first dimensionof the multi-dimensional liquid chromatography system is ahigh-performance liquid chromatography subsystem adapted formobile-phase temperature gradient elution chromatography.
 23. The methodof claim 1, wherein the first dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for reverse phase chromatography.
 24. The method ofclaim 1, wherein the first dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for adsorption chromatography.
 25. The method of claim1, wherein the first dimension of the multi-dimensional liquidchromatography system is a high-performance liquid chromatographysubsystem adapted for determining compositional variations offirst-dimension separated sample components, and the second dimension ofthe multi-dimensional liquid chromatography system is a high-performanceliquid chromatography subsystem adapted for size exclusionchromatography.
 26. The method of claim 1, wherein the polymer sample isa non-biological polymer sample.
 27. The method of claim 1, wherein thepolymer sample is a biological polymer sample.
 28. The method of claim1, wherein the polymer sample being characterized is a member of alibrary of polymer samples comprising four or more different polymersamples, the method further comprising: repeating the steps of injectinga polymer sample into the first-dimension mobile phase, separating acomponent of the polymer sample in the first-dimension liquidchromatography column, sampling the first-dimension mobile phase eluent,injecting sampled volumes in to the second-dimension mobile phase,separating a subcomponent of sampled portions of the first-dimensionseparated components in the second-dimension liquid chromatographycolumn, and detecting a property of the second-dimension separatedsubcomponents for each of the polymer samples of the library.
 29. Themethod of claim 1, wherein the polymer sample is a polymerizationproduct mixture that is untreated or pretreated only with one or moresteps selected from the group consisting of non-chromatographicseparation, dilution, mixing, and redissolution.
 30. A method forcharacterizing a polymer sample in a multi-dimensional liquidchromatography system, the method comprising: injecting the polymersample into a first-dimension mobile phase of a first dimension of themulti-dimensional liquid chromatography system, wherein the firstdimension of the multi-dimensional liquid chromatography system is ahigh-performance liquid chromatography subsystem adapted for determiningcompositional variations of first-dimension separated sample components;chromatographically separating at least one sample component of theinjected polymer sample from other sample components thereof in afirst-dimension liquid chromatography column, such that afirst-dimension mobile phase eluent from the first-dimension columncomprises two or more first-dimension separated sample components;sampling discrete volumes of the first-dimension mobile phase eluent atregularly recurring time intervals, such that at least a portion of eachof the first-dimension separated sample components are sampled;injecting each of the sampled volumes of the first-dimension mobilephase eluent directly into a second-dimension mobile phase of the seconddimension of the multi-dimensional liquid chromatography system, whereinthe second dimension of the multi-dimensional liquid chromatographysystem is a high-performance liquid chromatography subsystem adapted forsize exclusion chromatography; chromatographically separating at leastone subcomponent of the sampled portions of each of the first-dimensionseparated sample components from other subcomponents thereof in asecond-dimension liquid chromatography column, such that asecond-dimension mobile phase eluent from the second-dimension columncomprises two or more second-dimension separated subcomponents for eachof the sampled portions of each of the first-dimension separated samplecomponents; and detecting a property of the second-dimension separatedsubcomponents in the second-dimension mobile phase eluent using aflow-through detector.
 31. The method of claim 30, wherein the firstdimension of the multi-dimensional liquid chromatography system is ahigh-performance liquid chromatography subsystem adapted formobile-phase compositional gradient elution chromatography.
 32. Themethod of claim 30, wherein the first dimension of the multi-dimensionalliquid chromatography system is a high-performance liquid chromatographysubsystem adapted for mobile-phase temperature gradient elutionchromatography.
 33. The method of claim 30, wherein the first dimensionof the multi-dimensional liquid chromatography system is ahigh-performance liquid chromatography subsystem adapted for reversephase chromatography.
 34. The method of claim 30, wherein the firstdimension of the multi-dimensional liquid chromatography system is ahigh-performance liquid chromatography subsystem adapted for adsorptionchromatography.
 35. A method for characterizing a polymer sample in amulti-dimensional liquid chromatography system, the method comprising:injecting the polymer sample into a first-dimension mobile phase of afirst dimension of the multi-dimensional liquid chromatography system,wherein the polymer sample is a member of a library of polymer samplescomprising four or more different polymer samples; chromatographicallyseparating at least one sample component of the injected polymer samplefrom other sample components thereof in a first-dimension liquidchromatography column, such that a first-dimension mobile phase eluentfrom the first-dimension column comprises two or more first-dimensionseparated sample components; sampling discrete volumes of thefirst-dimension mobile phase eluent at regularly recurring timeintervals, such that at least a portion of each of the first-dimensionseparated sample components are sampled; injecting each of the sampledvolumes of the first-dimension mobile phase eluent directly into asecond-dimension mobile phase of the second dimension of themulti-dimensional liquid chromatography system; chromatographicallyseparating at least one subcomponent of the sampled portions of each ofthe first-dimension separated sample components from other subcomponentsthereof in a second-dimension liquid chromatography column, such that asecond-dimension mobile phase eluent from the second-dimension columncomprises two or more second-dimension separated subcomponents for eachof the sampled portions of each of the first-dimension separated samplecomponents; detecting a property of the second-dimension separatedsubcomponents in the second-dimension mobile phase eluent using aflow-through detector; and repeating the steps of injecting a polymersample into the first-dimension mobile phase, separating a component ofthe polymer sample in the first-dimension liquid chromatography column,sampling the first-dimension mobile phase eluent, injecting sampledvolumes in to the second-dimension mobile phase, separating asubcomponent of sampled portions of the first-dimension separatedcomponents in the second-dimension liquid chromatography column, anddetecting a property of the second-dimension separated subcomponents foreach of the polymer samples of the library.
 36. The method of claim 35,wherein the polymer samples of the library are non-biological polymersamples.
 37. The method of claim 35, wherein the library comprises fouror more different non-biological polymers on a common substrate.
 38. Themethod of claim 35, wherein the library comprises four or more differentnon-biological polymers synthesized in a parallel reaction vessel. 39.The method of claim 35, wherein the four or more differentnon-biological polymer samples are polymerization product mixturesresulting from parallel polymerization reactions that are varied withrespect to a factor affecting polymerization.
 40. The method of claim35, wherein the four or more different non-biological polymer samplesare polymerization product mixtures resulting from parallelpolymerization reactions that are varied with respect to one or more ofreactant materials, catalysts, catalysts precursors, initiators,additives, or the relative amounts thereof.
 41. The method of claim 35,wherein the four or more different non-biological polymer samples arepolymerization product mixtures resulting from parallel polymerizationreactions that are varied with respect to reaction conditions.